Protein formulations and methods of making same

ABSTRACT

The invention provides an aqueous formulation comprising water and a protein, and methods of making the same. The aqueous formulation of the invention may be a high protein formulation and/or may have low levels of conductivity resulting from the low levels of ionic excipients. Also included in the invention are formulations comprising water and proteins having low osmolality.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/228,478 filed on Dec. 20, 2018, pending, which is a continuation ofU.S. patent application Ser. No. 15/423,503 filed on Feb. 2, 2017,abandoned, which is a continuation of U.S. patent application Ser. No.15/096,043, filed on Apr. 11, 2016, abandoned, which is a continuationof U.S. patent application Ser. No. 14/796,389, filed on Jul. 10, 2015,abandoned, which is a divisional of U.S. patent application Ser. No.14/506,576, filed on Oct. 3, 2014, now U.S. Pat. No. 9,085,619, issuedon Jul. 21, 2015, which is a continuation of U.S. patent applicationSer. No. 13/774,735, now U.S. Pat. No. 8,883,146, issued on Nov. 11,2014, which is a continuation of U.S. patent application Ser. No.12/325,049, now U.S. Pat. No. 8,420,081, issued on Apr. 16, 2013, whichclaims the benefit of priority to U.S. Provisional Application No.61/004,992, filed on Nov. 30, 2007. The entire contents of each of theforegoing applications are hereby incorporated by reference herein.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Dec. 28, 2020, isnamed 13371-219-999_SEQLIST.txt and 4638 bytes in size, and is identicalto the paper copy to the Sequence Listing.

BACKGROUND OF THE INVENTION

A basic principle of pharmaceutical protein formulations is that certaininstabilities must be overcome. Degradation pathways of proteins can beseparated into two distinct classes, involving chemical instability andphysical instability. Chemical instabilities lead to the modification ofthe protein through bond formation or cleavage. Examples of chemicalinstability problems include deamidation, racemization, hydrolysis,oxidation, beta elimination and disulfide exchange. Physicalinstabilities, on the other hand, do not lead to covalent changes inproteins. Rather, they involve changes in the higher order structure(secondary and above) of proteins. These include denaturation,adsorption to surfaces, aggregation and precipitation (Manning et al.,Pharm. Res. 6, 903 (1989)).

It is generally accepted that these instabilities, which can have greateffect on the commercial viability and efficacy of pharmaceuticalprotein formulations, can be overcome by including additional moleculesin the formulation. Protein stability can be improved by includingexcipients that interact with the protein in solution to keep theprotein stable, soluble and unaggregated. For example, salt compoundsand other ionic species are very common additives to proteinformulations. They assist in fighting denaturation of proteins bybinding to proteins in a non-specific fashion and increasing thermalstability. Salt compounds (e.g., NaCl, KCl) have been used successfullyin commercial insulin preparations to fight aggregation andprecipitation (ibid. at 911). Amino acids (e.g., histidine, arginine)have been shown to reduce alterations in proteins' secondary structureswhen used as formulation additives (Tian et al., Int'l J. Pharm. 355, 20(2007)). Other examples of commonly used additives include polyalcoholmaterials such as glycerol and sugars, and surfactants such asdetergents, both nonionic (e.g., Tween, Pluronic) and anionic (sodiumdodecyl sulfate). The near universal prevalence of additives in allliquid commercial protein formulations indicates that protein solutionswithout such compounds may encounter challenges with degradation due toinstabilities.

The primary goal of protein formulation is to maintain the stability ofa given protein in its native, pharmaceutically active form overprolonged periods of time to guarantee acceptable shelf-life of thepharmaceutical protein drug. Maintaining the stability and solubility ofproteins in solution, however, is especially challenging inpharmaceutical formulations where the additives are included intotherapeutics. To date, biologic formulations require additionalexcipients to maintain protein stability. Typically, liquidpharmaceutical formulations contain multiple additives for stability.For example, a liquid formulation for patient self-administration ofhuman growth hormone, Norditropin SimpleXx®, contains the additivesmannitol (a sugar alcohol), histidine and poloxamer 188 (a surfactant)to stabilize the hormone.

Pharmaceutical additives need to be soluble, non-toxic and used atparticular concentrations that provide stabilizing effects on thespecific therapeutic protein. Since the stabilizing effects of additivesare protein- and concentration-dependent, each additive being consideredfor use in a pharmaceutical formulation must be carefully tested toensure that it does not cause instability or have other negative effectson the chemical or physical make-up of the formulation. Ingredients usedto stabilize the protein may cause problems with protein stability overtime or with protein stability in changing environments during storage.

Typically, long shelf-life is achieved by storing the protein in frozenfrom (e.g., at −80° C.) or by subjecting the protein to a lyophilizationprocess, i.e., by storing the protein in lyophilized form, necessitatinga reconstitution step immediately before use and thus posing asignificant disadvantage with regard to patient convenience. However,freezing a protein formulation for storage may lead to localized highconcentrations of proteins and additives, which can create localextremes in pH, degradation and protein aggregation within theformulation. In addition, it is well known to those skilled in the artthat freezing and thawing processes often impact protein stability,meaning that even storage of the pharmaceutical protein in frozen formcan be associated with the loss of stability due to the freezing andthawing step. Also, the first process step of lyophilization involvesfreezing, which can negatively impact protein stability. In industrysettings, a pharmaceutical protein may be subjected to repeatedfreeze-thaw processing during Drug Substance manufacturing (holdingsteps, storage, re-freeze and re-thaw to increase timing and batch sizeflexibility in Drug Product fill-finishing) and during subsequent DrugProduct fill-finishing (lyophilization). Since it is well known that therisk of encountering protein instability phenomena increases withincreasing the number of freeze-thaw cycles a pharmaceutical proteinencounters, achieving formulation conditions that maintain proteinstability over repeated freeze-thaw processes is a challenging task.There is a need in the biopharmaceutical industry for formulations thatcan be frozen and thawed without creating undesired properties in theformulations, especially gradients of pH, osmolarity, density or proteinor excipient concentration.

Often protein-based pharmaceutical products need to be formulated athigh concentrations for therapeutic efficacy. Highly concentratedprotein formulations are desirable for therapeutic uses since they allowfor dosages with smaller volumes, limiting patient discomfort, and aremore economically packaged and stored. The development of high proteinconcentration formulations, however, presents many challenges, includingmanufacturing, stability, analytical, and, especially for therapeuticproteins, delivery challenges. For example, difficulties with theaggregation, insolubility and degradation of proteins generally increaseas protein concentrations in formulations are raised (for review, seeShire, S. J. et al. J. Pharm. Sci., 93, 1390 (2004)). Previously unseennegative effects may be caused by additives that, at lowerconcentrations of the additives or the protein, provided beneficialeffects. The production of high concentration protein formulations maylead to significant problems with opalescence, aggregation andprecipitation. In addition to the potential for nonnative proteinaggregation and particulate formation, reversible self-association mayoccur, which may result in increased viscosity or other properties thatcomplicate delivery by injection. High viscosity also may complicatemanufacturing of high protein concentrations by filtration approaches.

Thus, pharmaceutical protein formulations typically carefully balanceingredients and concentrations to enhance protein stability andtherapeutic requirements while limiting any negative side-effects.Biologic formulations should include stable protein, even at highconcentrations, with specific amounts of excipients reducing potentialtherapeutic complications, storage issues and overall cost.

As proteins and other biomacromolecules gain increased interest as drugmolecules, formulations for delivering such molecules are becoming animportant issue. Despite the revolutionary progress in the large-scalemanufacturing of proteins for therapeutic use, effective and convenientdelivery of these agents in the body remains a major challenge due totheir intrinsic physicochemical and biological properties, includingpoor permeation through biological membranes, large molecular size,short plasma half life, self association, physical and chemicalinstability, aggregation, adsorption, and immunogenicity.

SUMMARY OF THE INVENTION

The invention is directed towards the surprising findings that proteinsformulated in water maintain solubility, as well as stability, even athigh concentrations, during long-term liquid storage or other processingsteps, such as freeze/thawing and lyophilization.

The present invention relates to methods and compositions for aqueousprotein formulations which comprise water and a protein, where theprotein is stable without the need for additional agents. Specifically,the methods and compositions of the invention are based on adiafiltration process wherein a first solution containing the protein ofinterest is diafiltered using water as a diafiltration medium. Theprocess is performed such that there is at least a determined volumeexchange, e.g., a five fold volume exchange, with the water. Byperforming the methods of the invention, the resulting aqueousformulation has a significant decrease in the overall percentage ofexcipients in comparison to the initial protein solution. For example,95-99% less excipients are found in the aqueous formulation incomparison to the initial protein solution. Despite the decrease inexcipients, the protein remains soluble and retains its biologicalactivity, even at high concentrations. In one aspect, the methods of theinvention result in compositions comprising an increase in concentrationof the protein while decreasing additional components, such as ionicexcipients. As such, the hydrodynamic diameter of the protein in theaqueous formulation is smaller relative to the same protein in astandard buffering solution, such as phosphate buffered saline (PBS).

The formulation of the invention has many advantages over standardbuffered formulations. In one aspect, the aqueous formulation compriseshigh protein concentrations, e.g., 50 to 200 mg/mL or more. Proteins ofall sizes may be included in the formulations of the invention, even atincreased concentrations. Despite the high concentration of protein, theformulation has minimal aggregation and can be stored using variousmethods and forms, e.g., freezing, without deleterious effects thatmight be expected with high protein formulations. Formulations of theinvention do not require excipients, such as, for example, surfactantsand buffering systems, which are used in traditional formulations tostabilize proteins in solution. As a result of the low level of ionicexcipients, the aqueous formulation of the invention has lowconductivity, e.g., less than 2 mS/cm. The methods and compositions ofthe invention also provide aqueous protein formulations having lowosmolality, e.g., no greater than 30 mOsmol/kg. In addition, theformulations described herein are preferred over standard formulationsbecause they have decreased immunogenicity due to the lack of additionalagents needed for protein stabilization.

The methods and compositions of the invention may be used to provide anaqueous formulation comprising water and any type of protein ofinterest. In one aspect, the methods and compositions of the inventionare used for large proteins, including proteins which are larger than 47kDa. Antibodies, and fragments thereof, including those used for in vivoand in vitro purposes, are another example of proteins which may be usedin the methods and compositions of the invention.

Furthermore, the multiple step purification and concentration processesthat are necessary to prepare proteins and peptide formulations oftenintroduce variability in compositions, such that the precise compositionof a formulation may vary from lot to lot. Federal regulations requirethat drug compositions be highly consistent in their formulationsregardless of the location of manufacture or lot number. Methods of theinvention can be used to create solutions of proteins formulated inwater to which buffers and excipients are added back in precise amounts,allowing for the creation of protein formulations with preciseconcentrations of buffers and/or excipients.

In one embodiment, the invention provides an aqueous formulationcomprising a protein and water, wherein the formulation has certaincharacteristics, such as, but not limited to, low conductivity, e.g., aconductivity of less than about 2.5 mS/cm, a protein concentration of atleast about 10 μg/mL, an osmolality of no more than about 30 mOsmol/kg,and/or the protein has a molecular weight (M_(w)) greater than about 47kDa. In one embodiment, the formulation of the invention has improvedstability, such as, but not limited to, stability in a liquid form foran extended time (e.g., at least about 3 months or at least about 12months) or stability through at least one freeze/thaw cycle (if not morefreeze/thaw cycles). In one embodiment, the formulation is stable for atleast about 3 months in a form selected from the group consisting offrozen, lyophilized, or spray-dried.

In one embodiment, proteins included in the formulation of the inventionmay have a minimal size, including, for example, a M_(w) greater thanabout 47 kDa, a M_(w) greater than about 57 kDa, a M_(w) greater thanabout 100 kDa, a M_(w) greater than about 150 kDa, a M_(w) greater thanabout 200 kDa, or a M_(w) greater than about 250 kDa.

In one embodiment, the formulation of the invention has a lowconductivity, including, for example, a conductivity of less than about2.5 mS/cm, a conductivity of less than about 2 mS/cm, a conductivity ofless than about 1.5 mS/cm, a conductivity of less than about 1 mS/cm, ora conductivity of less than about 0.5 mS/cm.

In one embodiment, proteins included in the formulation of the inventionhave a given concentration, including, for example, a concentration ofat least about 1 mg/mL, at least about 10 mg/mL, at least about 50mg/mL, at least about 100 mg/mL, at least about 150 mg/mL, at leastabout 200 mg/mL, or greater than about 200 mg/mL.

In one embodiment, the formulation of the invention has an osmolality ofno more than about 15 mOsmol/kg.

In one embodiment, the invention provides an aqueous formulationcomprising water and a given concentration of a protein, wherein theprotein has a hydrodynamic diameter (D_(h)) which is at least about 50%less than the D_(h) of the protein in a buffered solution at the givenconcentration. In one embodiment, the D_(h) of the protein is at leastabout 50% less than the D_(h) of the protein in phosphate bufferedsaline (PBS) at the given concentration; the D_(h) of the protein is atleast about 60% less than the D_(h) of the protein in PBS at the givenconcentration; the D_(h) of the protein is at least about 70% less thanthe D_(h) of the protein in PBS at the given concentration.

In one embodiment, the invention provides an aqueous formulationcomprising a protein, such as, but not limited to, an antibody, or anantigen-binding fragment, wherein the protein has a hydrodynamicdiameter (D_(h)) of less than about 5 In one embodiment, the protein hasa D_(h) of less than about 3 μm.

Any protein may be used in the methods and compositions of theinvention. In one embodiment, the formulation comprises a therapeuticprotein. In one embodiment, the formulation comprises an antibody, or anantigen-binding fragment thereof. Types of antibodies, or antigenbinding fragments, that may be included in the methods and compositionsof the invention include, but are not limited to, a chimeric antibody, ahuman antibody, a humanized antibody, and a domain antibody (dAb). Inone embodiment, the antibody, or antigen-binding fragment thereof, is ananti-TNFα, such as but not limited to adalimumab or golimumab, or ananti-IL-12 antibody, such as but not limited to J695. In addition, theformulation of the invention may also include at least two distincttypes of proteins, e.g., adalimumab and J695.

In yet another embodiment of the invention, the formulation may furthercomprise a non-ionizable excipient. Examples of non-ionizable excipientsinclude, but are not limited to, a sugar alcohol or polyol (e.g,mannitol or sorbitol), a non-ionic surfactant (e.g., polysorbate 80,polysorbate 20, polysorbate 40, polysorbate 60), and/or a sugar (e.g,sucrose). Other non-limiting examples of non-ionizable excipients thatmay be further included in the formulation of the invention include, butare not limited to, non-trehalose, raffinose, and maltose.

In one embodiment, the formulation does not comprise an agent selectedfrom the group consisting of a tonicity modifier, a stabilizing agent, asurfactant, an anti-oxidant, a cryoprotectant, a bulking agent, alyroprotectant, a basic component, and an acidic component.

The formulation of the invention may be suitable for any use, includingboth in vitro and in vivo uses. In one embodiment, the formulation ofthe invention is suitable for administration to a subject via a mode ofadministration, including, but not limited to, subcutaneous,intravenous, inhalation, intradermal, transdermal, intraperitoneal, andintramuscular administration. The formulation of the invention may beused in the treatment of a disorder in a subject.

Also included in the invention are devices that may be used to deliverthe formulation of the invention. Examples of such devices include, butare not limited to, a syringe, a pen, an implant, a needle-freeinjection device, an inhalation device, and a patch.

In one embodiment, the formulation of the invention is a pharmaceuticalformulation.

The invention also provides a method of preparing an aqueous formulationcomprising a protein and water, the method comprising providing theprotein in a first solution, and subjecting the first solution todiafiltration using water as a diafiltration medium until at least afive fold volume exchange with the water has been achieved to therebyprepare the aqueous formulation. In one embodiment, the protein in theresulting formulation retains its biological activity.

The invention further provides a method of preparing an aqueousformulation of a protein, the method comprising providing the protein ina first solution; subjecting the first solution to diafiltration usingwater as a diafiltration medium until at least a five-fold volumeexchange with the water has been achieved to thereby prepare adiafiltered protein solution; and concentrating the diafiltered proteinsolution to thereby prepare the aqueous formulation of the protein. Inone embodiment, the protein in the resulting formulation retains itsbiological activity.

In one embodiment, the concentration of the diafiltered protein solutionis achieved via centrifugation.

In one embodiment, the diafiltration medium consists of water.

In one embodiment, the first solution is subjected to diafiltration withwater until a volume exchange greater than a five-fold volume exchangeis achieved. In one embodiment, the first solution is subjected todiafiltration with water until at least about a six-fold volume exchangeis achieved. In one embodiment, the first solution is subjected todiafiltration with water until at least about a seven-fold volumeexchange is achieved.

In one embodiment, the aqueous formulation has a final concentration ofexcipients which is at least about 95% less than the first solution.

In one embodiment, the aqueous formulation has a final concentration ofexcipients which is at least about 99% less than the first solution.

In one embodiment, the first protein solution is obtained from amammalian cell expression system and has been purified to remove hostcell proteins (HCPs).

In one embodiment, the method of the invention further comprises addingan excipient to the aqueous formulation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the SEC chromatogram of Adalimumab reference standardAFP04C (bottom line), Adalimumab DS (drug substance before (middle line)and after DF/UF processing (top line).

FIG. 2 shows the impact of sorbitol (a non-ionizable excipient) and NaCl(ionizable excipient) concentrations on the hydrodynamic diameter (Dh)of Adalimumab monomer upon addition of the excipient compound toDF/UF-processed Adalimumab monomer.

FIG. 3 shows the IEC profile of J695 reference standard (bottom graph)and J695 DS, pH adjusted to pH 4.4 (top graph).

FIG. 4 shows the IEC profile of J695 after DF/UF with Milli-Q water, pH4.7 (top graph), and J695 DS before DF/UF, pH adjusted to pH 4.4 (bottomcurve).

FIG. 5 graphically depicts the correlation of hydrodynamic diameter(z-average) and concentration of Adalimumab (dissolved in WFI). X:determined with an SOP using 1.1 mPas as assumed sample viscosity. y:determined with an SOP using 1.9 mPas as assumed sample viscosity.

FIG. 6 graphically depicts the correlation of hydrodynamic diameter(peak monomer) and concentration of Adalimumab (dissolved in WFI). X:determined with an SOP using 1.1 mPas as assumed sample viscosity. y:determined with an SOP using 1.9 mPas as assumed sample viscosity

FIG. 7 graphically depicts the correlation of hydrodynamic diameter(z-average) and concentration of J695 (dissolved in WFI). X: determinedwith an SOP using 1.1 mPas as assumed sample viscosity: determined withan SOP using 1.9 mPas as assumed sample viscosity

FIG. 8 graphically depicts the correlation of hydrodynamic diameter(peak monomer) and concentration of J695 (dissolved in WFI). X:determined with an SOP using 1.1 mPas as assumed sample viscosity. y:determined with an SOP using 1.9 mPas as assumed sample viscosity.

FIG. 9 shows the sum of lysine 0, 1 and 2 of Adalimumab [%] independence on Adalimumab concentration in water for injection.

FIG. 10 shows the sum of peak 1 to 7 of J695 [%] in dependence on J695concentration in water for injection.

FIG. 11 shows the sum of acidic peaks of J695 [%] in dependence on J695concentration in water for injection.

FIG. 12 shows the sum of basic peaks of J695 [%] in dependence on J695concentration in water for injection (WFI).

FIG. 13 shows the efficiency of the dialysis performed in Example 12, interms of the reduction of components responsible for osmolality andconductivity of the formulation (BDS, 74 mg/ml, 10 ml sample volume,SpectraPor7 MWCO10 k).

FIG. 14 shows the stability of pH levels in dialyzed Adalimumab BulkSolutions. pH levels before and after dialysis against deionized water(1:1,000,000) are shown. (BDS, 74 mg/ml, 10 ml sample volume,SpectraPor7 MWCO10 k)

FIG. 15 shows bottle mapping density data for 250 mg/ml and 200 mg/mllow-ionic Adalimumab solutions after freeze thaw.

FIG. 16 shows bottle mapping pH data for 250 mg/ml and 200 mg/mllow-ionic Adalimumab solutions after freeze thaw.

FIG. 17 shows bottle mapping concentration data for 250 mg/ml and 200mg/ml low-ionic Adalimumab solutions after freeze thaw.

FIG. 18 shows bottle mapping osmolality data for 250 mg/ml and 200 mg/mllow-ionic Adalimumab solutions after freeze thaw.

FIG. 19 shows bottle mapping conductivity data for 250 mg/ml and 200mg/ml low-ionic Adalimumab solutions after freeze thaw.

FIG. 20 shows SEC analysis of low-ionic Adalimumab (referred to as D2E7in FIG. 20) solutions that were either stored at 2-8° C. for 8.5 monthsafter DF/UF (bottom curve) or stored at −80° C. for 4.5 months afterDF/UF (top curve).

FIG. 21 shows the stability of the monoclonal antibody 1D4.7 formulatedin various solutions and in water before freeze-thaw procedures (T0) andafter each of four freeze-thaws (T1, T2, T3 and T4).

FIG. 22 shows the stability of the monoclonal antibody 13C5.5 formulatedin water and with various buffers before freeze-thaw procedures (T0) andafter each of four freeze-thaws (T1, T2, T3 and T4). Blank=WFI controlsample.

FIG. 23 shows the stability of the monoclonal antibody 13C5.5 formulatedin water and with various excipients added, before freeze-thawprocedures (T0) and after each of four freeze-thaws (T1, T2, T3 and T4).Blank=WFI control sample.

FIG. 24 shows the impact of the concentration of Adalimumab (WFIformulation) and solution pH on solution viscosity.

FIG. 25 shows turbidity data for Adalimumab solutions (WFI formulations)of various concentrations and pH values.

FIG. 26 shows hydrodynamic diameter (Dh) data for Adalimumab solutions(WFI formulations) at various pH values and concentrations.

FIG. 27 shows a size distribution by intensity graph (Dh measurements)for Adalimumab in water solutions, pH 5, at various concentrations.

FIG. 28 shows size distribution by intensity for 100 mg/mL Adalimumab inwater at various pH levels.

FIG. 29 also shows size distribution by intensity for 100 mg/mLAdalimumab in water at various pH levels.

FIG. 30 shows monomer content (SEC) for Adalimumab in water.

FIG. 31 shows aggregate content (SEC) for Adalimumab in water.

FIG. 32 shows the viscosity of two J695 solutions (WFI formulations) asa function of solution temperature.

FIG. 33 graphically depicts 1D4.7 antibody stability as measured bysubvisible particle (>1 μm) during repeated freeze/thaw (f/t) cycles fora number of different formulations.

FIG. 34 graphically depicts 13C5.5 antibody stability as measured bysubvisible particle (>10 μm) during repeated freeze/thaw (f/t) cyclesfor a number of different formulations.

FIG. 35 graphically depicts 13C5.5 antibody stability as measured bysubvisible particle (>1 μm) during repeated freeze/thaw (f/t) cycles fora number of different formulations.

FIG. 36 graphically depicts 7C6 antibody stability as measured bysubvisible particle (>1 μm) during repeated freeze/thaw (f/t) cycles fora number of different formulations.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

In order that the present invention may be more readily understood,certain terms are first defined.

As used herein, the term “acidic component” refers to an agent,including a solution, having an acidic pH, i.e., less than 7.0. Examplesof acidic components include phosphoric acid, hydrochloric acid, aceticacid, citric acid, oxalic acid, succinic acid, tartaric acid, lacticacid, malic acid, glycolic acid and fumaric acid. In one embodiment, theaqueous formulation of the invention does not include an acidiccomponent.

As used herein, the term “antioxidant” is intended to mean an agentwhich inhibits oxidation and thus is used to prevent the deteriorationof preparations by the oxidative process. Such compounds include by wayof example and without limitation, acetone, sodium bisulfate, ascorbicacid, ascorbyl palmitate, citric acid, butylated hydroxyanisole,butylated hydroxytoluene, hydrophosphorous acid, monothioglycerol,propyl gallate, methionine, sodium ascorbate, sodium citrate, sodiumsulfide, sodium sulfite, sodium bisulfate, sodium formaldehydesulfoxylate, thioglycolic acid, sodium metabisulfite, EDTA (edetate),pentetate and others known to those of ordinary skill in the art.

The term “aqueous formulation” refers to a solution in which the solventis water.

As used herein, the term “basic component” refers to an agent which isalkaline, i.e., pH greater than 7.0. Examples of basic componentsinclude potassium hydroxide (KOH) and sodium hydroxide (NaOH)

As used herein, the term “bulking agent” is intended to mean a compoundused to add bulk to the reconstitutable solid and/or assist in thecontrol of the properties of the formulation during preparation. Suchcompounds include, by way of example and without limitation, dextran,trehalose, sucrose, polyvinylpyrrolidone, lactose, inositol, sorbitol,dimethylsulfoxide, glycerol, albumin, calcium lactobionate, and othersknown to those of ordinary skill in the art.

The term “conductivity,” as used herein, refers to the ability of anaqueous solution to conduct an electric current between two electrodes.Generally, electrical conductivity or specific conductivity is a measureof a material's ability to conduct an electric current. In solution, thecurrent flows by ion transport. Therefore, with an increasing amount ofions present in the aqueous solution, the solution will have a higherconductivity. The unit of measurement for conductivity is mmhos (mS/cm),and can be measured using a conductivity meter sold, e.g., by OrionResearch, Inc. (Beverly, Mass.). The conductivity of a solution may bealtered by changing the concentration of ions therein. For example, theconcentration of ionic excipients in the solution may be altered inorder to achieve the desired conductivity.

The term “cryoprotectants” as used herein generally includes agents,which provide stability to the protein from freezing-induced stresses.Examples of cryoprotectants include polyols such as, for example,mannitol, and include saccharides such as, for example, sucrose, as wellas including surfactants such as, for example, polysorbate, poloxamer orpolyethylene glycol, and the like. Cryoprotectants also contribute tothe tonicity of the formulations.

As used herein, the terms “ultrafiltration” or “UF” refers to anytechnique in which a solution or a suspension is subjected to asemi-permeable membrane that retains macromolecules while allowingsolvent and small solute molecules to pass through. Ultrafiltration maybe used to increase the concentration of macromolecules in a solution orsuspension. In a preferred embodiment, ultrafiltration is used toincrease the concentration of a protein in water.

As used herein, the term “diafiltration” or “DF” is used to mean aspecialized class of filtration in which the retentate is diluted withsolvent and re-filtered, to reduce the concentration of soluble permeatecomponents. Diafiltration may or may not lead to an increase in theconcentration of retained components, including, for example, proteins.For example, in continuous diafiltration, a solvent is continuouslyadded to the retentate at the same rate as the filtrate is generated. Inthis case, the retentate volume and the concentration of retainedcomponents does not change during the process. On the other hand, indiscontinuous or sequential dilution diafiltration, an ultrafiltrationstep is followed by the addition of solvent to the retentate side; ifthe volume of solvent added to the retentate side is not equal orgreater to the volume of filtrate generated, then the retainedcomponents will have a high concentration. Diafiltration may be used toalter the pH, ionic strength, salt composition, buffer composition, orother properties of a solution or suspension of macromolecules.

As used herein, the terms “diafiltration/ultrafiltration” or “DF/UF”refer to any process, technique or combination of techniques thataccomplishes ultrafiltration and/or diafiltration, either sequentiallyor simultaneously.

As used herein, the term “diafiltration step” refers to a total volumeexchange during the process of diafiltration.

The term “excipient” refers to an agent that may be added to aformulation to provide a desired consistency, (e.g., altering the bulkproperties), to improve stability, and/or to adjust osmolality. Examplesof commonly used excipients include, but are not limited to, sugars,polyols, amino acids, surfactants, and polymers. The term “ionicexcipient” or “ionizable excipient,” as used interchangeably herein,refers to an agent that has a net charge. In one embodiment, the ionicexcipient has a net charge under certain formulation conditions, such aspH. Examples of an ionic excipient include, but are not limited to,histidine, arginine, and sodium chloride. The term “non-ionic excipient”or “non-ionizable excipient,” as used interchangeably herein, refers toan agent having no net charge. In one embodiment, the non-ionicexcipient has no net charge under certain formulation conditions, suchas pH. Examples of non-ionic excipients include, but are not limited to,sugars (e.g., sucrose), sugar alcohols (e.g., mannitol), and non-ionicsurfactants (e.g., polysorbate 80).

The term “first protein solution” or “first solution” as used herein,refers to the initial protein solution or starting material used in themethods of the invention, i.e., the initial protein solution which isdiafiltered into water. In one embodiment, the first protein solutioncomprises ionic excipients, non-ionic excipients, and/or a bufferingsystem.

The term “hydrodynamic diameter” or “D_(h)” of a particle refers to thediameter of a sphere that has the density of water and the same velocityas the particle. Thus the term “hydrodynamic diameter of a protein” asused herein refers to a size determination for proteins in solutionusing dynamic light scattering (DLS). A DLS-measuring instrumentmeasures the time-dependent fluctuation in the intensity of lightscattered from the proteins in solution at a fixed scattering angle.Protein Dh is determined from the intensity autocorrelation function ofthe time-dependent fluctuation in intensity. Scattering intensity dataare processed using DLS instrument software to determine the value forthe hydrodynamic diameter and the size distribution of the scatteringmolecules, i.e. the protein specimen.

The term “lyoprotectant” as used herein includes agents that providestability to a protein during water removal during the drying orlyophilisation process, for example, by maintaining the properconformation of the protein. Examples of lyoprotectants includesaccharides, in particular di- or trisaccharides. Cryoprotectants mayalso provide lyoprotectant effects.

The term “pharmaceutical” as used herein with reference to acomposition, e.g., an aqueous formulation, that it is useful fortreating a disease or disorder.

The term “protein” is meant to include a sequence of amino acids forwhich the chain length is sufficient to produce the higher levels ofsecondary and/or tertiary and/or quaternary structure. This is todistinguish from “peptides” or other small molecular weight drugs thatdo not have such structure. In one embodiment, the proteins used hereinhave a molecular weight of at least about 47 kD. Examples of proteinsencompassed within the definition used herein include therapeuticproteins. A “therapeutically active protein” or “therapeutic protein”refers to a protein which may be used for therapeutic purposes, i.e.,for the treatment of a disorder in a subject. It should be noted thatwhile therapeutic proteins may be used for treatment purposes, theinvention is not limited to such use, as said proteins may also be usedfor in vitro studies. In a preferred embodiment, the therapeutic proteinis a fusion protein or an antibody, or antigen-binding portion thereof.In one embodiment, the methods and compositions of the inventioncomprise at least two distinct proteins, which are defined as twoproteins having distinct amino acid sequences. Additional distinctproteins do not include degradation products of a protein.

The phrase “protein is dissolved in water” as used herein refers to aformulation of a protein wherein the protein is dissolved in an aqueoussolution in which the amount of small molecules (e.g., buffers,excipients, salts, surfactants) has been reduced by DF/UF processing.Even though the total elimination of small molecules cannot be achievedin an absolute sense by DF/UF processing, the theoretical reduction ofexcipients achievable by applying DF/UF is sufficiently large to createa formulation of the protein essentially in water exclusively. Forexample, with 6 volume exchanges in a continuous mode DF/UF protocol,the theoretical reduction of excipients is ˜99.8% (ci=e^(−x), with cibeing the initial excipient concentration, and x being the number ofvolume exchanges).

The term “pharmaceutical formulation” refers to preparations which arein such a form as to permit the biological activity of the activeingredients to be effective, and, therefore may be administered to asubject for therapeutic use.

A “stable” formulation is one in which the protein therein essentiallyretains its physical stability and/or chemical stability and/orbiological activity upon storage. Various analytical techniques formeasuring protein stability are available in the art and are reviewed inPeptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., MarcelDekker, Inc., New York, N.Y., Pubs. (1991) and Jones, A. Adv. DrugDelivery Rev. 10: 29-90 (1993), for example. In one embodiment, thestability of the protein is determined according to the percentage ofmonomer protein in the solution, with a low percentage of degraded(e.g., fragmented) and/or aggregated protein. For example, an aqueousformulation comprising a stable protein may include at least 95% monomerprotein. Alternatively, an aqueous formulation of the invention mayinclude no more than 5% aggregate and/or degraded protein.

The term “stabilizing agent” refers to an excipient that improves orotherwise enhances stability. Stabilizing agents include, but are notlimited to, α-lipoic acid, α-tocopherol, ascorbyl palmitate, benzylalcohol, biotin, bisulfites, boron, butylated hydroxyanisole (BHA),butylated hydroxytoluene (BHT), ascorbic acid and its esters,carotenoids, calcium citrate, acetyl-L-camitine, chelating agents,chondroitin, chromium, citric acid, coenzyme Q-10, cysteine, cysteinehydrochloride, 3-dehydroshikimic acid (DHS), EDTA(ethylenediaminetetraacetic acid; edetate disodium), ferrous sulfate,folic acid, fumaric acid, alkyl gallates, garlic, glucosamine, grapeseed extract, gugul, magnesium, malic acid, metabisulfite, N-acetylcysteine, niacin, nicotinomide, nettle root, ornithine, propyl gallate,pycnogenol, saw palmetto, selenium, sodium bisulfite, sodiummetabisulfite, sodium sulfite, potassium sulfite, tartaric acid,thiosulfates, thioglycerol, thiosorbitol, tocopherol and their esters,e.g., tocopheral acetate, tocopherol succinate, tocotrienal,d-α-tocopherol acetate, vitamin A and its esters, vitamin B and itsesters, vitamin C and its esters, vitamin D and its esters, vitamin Eand its esters, e.g., vitamin E acetate, zinc, and combinations thereof.

The term “surfactants” generally includes those agents that protect theprotein from air/solution interface-induced stresses andsolution/surface induced-stresses. For example surfactants may protectthe protein from aggregation. Suitable surfactants may include, e.g.,polysorbates, polyoxyethylene alkyl ethers such as Brij 35®, orpoloxamer such as Tween 20, Tween 80, or poloxamer 188. Preferreddetergents are poloxamers, e.g., Poloxamer 188, Poloxamer 407;polyoxyethylene alkyl ethers, e.g., Brij 35®, Cremophor A25, SympatensALM/230; and polysorbates/Tweens, e.g., Polysorbate 20, Polysorbate 80,and Poloxamers, e.g., Poloxamer 188, and Tweens, e.g., Tween 20 andTween 80.

As used herein, the term “tonicity modifier” is intended to mean acompound or compounds that can be used to adjust the tonicity of aliquid formulation. Suitable tonicity modifiers include glycerin,lactose, mannitol, dextrose, sodium chloride, magnesium sulfate,magnesium chloride, sodium sulfate, sorbitol, trehalose, sucrose,raffinose, maltose and others known to those or ordinary skill in theart. In one embodiment, the tonicity of the liquid formulationapproximates that of the tonicity of blood or plasma.

The term “water” is intended to mean water that has been purified toremove contaminants, usually by distillation or reverse osmosis, alsoreferred to herein as “pure water”. In a preferred embodiment, waterused in the methods and compositions of the invention is excipient-free.In one embodiment, water includes sterile water suitable foradministration to a subject. In another embodiment, water is meant toinclude water for injection (WFI). In one embodiment, water refers todistilled water or water which is appropriate for use in in vitroassays. In a preferred embodiment, diafiltration is performed inaccordance with the methods of the invention using water alone as thediafiltration medium.

The term “antibody” as referred to herein includes whole antibodies andany antigen binding fragment (i.e., “antigen-binding portion”) or singlechains thereof. An “antibody” refers to a glycoprotein comprising atleast two heavy (H) chains and two light (L) chains inter-connected bydisulfide bonds, or an antigen binding portion thereof. Each heavy chainis comprised of a heavy chain variable region (abbreviated herein asV_(H)) and a heavy chain constant region. The heavy chain constantregion is comprised of three domains, CH1, CH2 and CH3. Each light chainis comprised of a light chain variable region (abbreviated herein asV_(L)) and a light chain constant region. The light chain constantregion is comprised of one domain, CL. The V_(H) and V_(L) regions canbe further subdivided into regions of hypervariability, termedcomplementarity determining regions (CDR), interspersed with regionsthat are more conserved, termed framework regions (FR). Each V_(H) andV_(L) is composed of three CDRs and four FRs, arranged fromamino-terminus to carboxy-terminus in the following order: FR1, CDR1,FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and lightchains contain a binding domain that interacts with an antigen. Theconstant regions of the antibodies may mediate the binding of theimmunoglobulin to host tissues or factors, including various cells ofthe immune system (e.g., effector cells) and the first component (Clq)of the classical complement system.

The term “antigen-binding portion” of an antibody (or simply “antibodyportion”), as used herein, refers to one or more fragments of anantibody that retain the ability to specifically bind to an antigen(e.g., TNFα, IL-12). It has been shown that the antigen-binding functionof an antibody can be performed by fragments of a full-length antibody.Examples of binding fragments encompassed within the term“antigen-binding portion” of an antibody include (i) a Fab fragment, amonovalent fragment consisting of the V_(L), V_(H), CL and CH1 domains;(ii) a F(ab′)₂ fragment, a bivalent fragment comprising two Fabfragments linked by a disulfide bridge at the hinge region; (iii) a Fdfragment consisting of the V_(H) and C_(H1) domains; (iv) a Fv fragmentconsisting of the V_(L) and V_(H) domains of a single arm of anantibody, (v) a dAb fragment (Ward et al, (1989) Nature 341:544-546),which consists of a V_(H) or V_(L) domain; and (vi) an isolatedcomplementarity determining region (CDR). Furthermore, although the twodomains of the Fv fragment, V_(L) and V_(H), are coded for by separategenes, they can be joined, using recombinant methods, by a syntheticlinker that enables them to be made as a single protein chain in whichthe VL and VH regions pair to form monovalent molecules (known as singlechain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; andHuston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Suchsingle chain antibodies are also intended to be encompassed within theterm “antigen-binding portion” of an antibody. These antibody fragmentsare obtained using conventional techniques known to those with skill inthe art, and the fragments are screened for utility in the same manneras are intact antibodies. In one embodiment of the invention, theantibody fragment is selected from the group consisting of a Fab, an Fd,an Fd′, a single chain Fv (scFv), an scFv_(a), and a domain antibody(dAb).

Still further, an antibody or antigen-binding portion thereof may bepart of a larger immunoadhesion molecule, formed by covalent ornoncovalent association of the antibody or antibody portion with one ormore other proteins or peptides. These other proteins or peptides canhave functionalities that allow for the purification of antibodies orantigen-binding portions thereof or allow for their association witheach other or other molecules. Thus examples of such immunoadhesionmolecules include use of the streptavidin core region to make atetrameric single chain variable fragment (scFv) molecules (Kipriyanovet al. (1995) Human Antibodies and Hybridomas 6:93-101) and the use of acysteine residue, a marker peptide and a C-terminal polyhistidine tag tomake bivalent and biotinylated scFv molecules (Kipriyanov et al. (1994)Mol. Immunol. 31:1047-1058). Antibody portions, such as Fab and F(ab′)₂fragments, can be prepared from whole antibodies using conventionaltechniques, such as papain or pepsin digestion, respectively, of wholeantibodies. Moreover, antibodies, antibody portions and immunoadhesionmolecules can be obtained using standard recombinant DNA techniques.

Two antibody domains are “complementary” where they belong to familiesof structures which form cognate pairs or groups or are derived fromsuch families and retain this feature. For example, a VH domain and a VLdomain of an antibody are complementary; two VH domains are notcomplementary, and two VL domains are not complementary. Complementarydomains may be found in other members of the immunoglobulin superfamily,such as the Vα and Vβ (or gamma and delta) domains of the T-cellreceptor.

The term “domain” refers to a folded protein structure which retains itstertiary structure independently of the rest of the protein. Generally,domains are responsible for discrete functional properties of proteins,and in many cases may be added, removed or transferred to other proteinswithout loss of function of the remainder of the protein and/or of thedomain. By single antibody variable domain is meant a folded polypeptidedomain comprising sequences characteristic of antibody variable domains.It therefore includes complete antibody variable domains and modifiedvariable domains, for example in which one or more loops have beenreplaced by sequences which are not characteristic of antibody variabledomains, or antibody variable domains which have been truncated orcomprise N- or C-terminal extensions, as well as folded fragments ofvariable domains which retain at least in part the binding activity andspecificity of the full-length domain.

Variable domains of the invention may be combined to form a group ofdomains; for example, complementary domains may be combined, such as VLdomains being combined with VH domains. Non-complementary domains mayalso be combined. Domains may be combined in a number of ways, involvinglinkage of the domains by covalent or non-covalent means.

A “dAb” or “domain antibody” refers to a single antibody variable domain(V_(H) or V_(L)) polypeptide that specifically binds antigen.

As used herein, the term “antigen binding region” or “antigen bindingsite” refers to the portion(s) of an antibody molecule, or antigenbinding portion thereof, which contains the amino acid residues thatinteract with an antigen and confers on the antibody its specificity andaffinity for the antigen.

The term “epitope” is meant to refer to that portion of any moleculecapable of being recognized by and bound by an antibody at one or moreof the antibody's antigen binding regions. In the context of the presentinvention, first and second “epitopes” are understood to be epitopeswhich are not the same and are not bound by a single monospecificantibody, or antigen-binding portion thereof.

The phrase “recombinant antibody” refers to antibodies that areprepared, expressed, created or isolated by recombinant means, such asantibodies expressed using a recombinant expression vector transfectedinto a host cell, antibodies isolated from a recombinant, combinatorialantibody library, antibodies isolated from an animal (e.g., a mouse)that is transgenic for human immunoglobulin genes (see e.g., Taylor etal. (1992) Nucl. Acids Res. 20:6287-6295) or antibodies prepared,expressed, created or isolated by any other means that involves splicingof particular immunoglobulin gene sequences (such as humanimmunoglobulin gene sequences) to other DNA sequences. Examples ofrecombinant antibodies include chimeric, CDR-grafted and humanizedantibodies.

The term “human antibody” refers to antibodies having variable andconstant regions corresponding to, or derived from, human germlineimmunoglobulin sequences as described by, for example, Kabat et al. (SeeKabat, et al. (1991) Sequences of Proteins of Immunological Interest,Fifth Edition, U.S. Department of Health and Human Services, NIHPublication No. 91-3242). The human antibodies of the invention,however, may include amino acid residues not encoded by human germlineimmunoglobulin sequences (e.g., mutations introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in vivo), forexample in the CDRs and in particular CDR3.

Recombinant human antibodies of the invention have variable regions, andmay also include constant regions, derived from human germlineimmunoglobulin sequences (See Kabat et al. (1991) Sequences of Proteinsof Immunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NIH Publication No. 91-3242). In certain embodiments,however, such recombinant human antibodies are subjected to in vitromutagenesis (or, when an animal transgenic for human Ig sequences isused, in vivo somatic mutagenesis) and thus the amino acid sequences ofthe VH and VL regions of the recombinant antibodies are sequences that,while derived from and related to human germline VH and VL sequences,may not naturally exist within the human antibody germline repertoire invivo. In certain embodiments, however, such recombinant antibodies arethe result of selective mutagenesis or backmutation or both.

The term “backmutation” refers to a process in which some or all of thesomatically mutated amino acids of a human antibody are replaced withthe corresponding germline residues from a homologous germline antibodysequence. The heavy and light chain sequences of a human antibody of theinvention are aligned separately with the germline sequences in theVBASE database to identify the sequences with the highest homology.Differences in the human antibody of the invention are returned to thegermline sequence by mutating defined nucleotide positions encoding suchdifferent amino acid. The role of each amino acid thus identified ascandidate for backmutation should be investigated for a direct orindirect role in antigen binding and any amino acid found after mutationto affect any desirable characteristic of the human antibody should notbe included in the final human antibody. To minimize the number of aminoacids subject to backmutation those amino acid positions found to bedifferent from the closest germline sequence but identical to thecorresponding amino acid in a second germline sequence can remain,provided that the second germline sequence is identical and colinear tothe sequence of the human antibody of the invention for at least 10,preferably 12 amino acids, on both sides of the amino acid in question.Backmutation may occur at any stage of antibody optimization.

The term “chimeric antibody” refers to antibodies which comprise heavyand light chain variable region sequences from one species and constantregion sequences from another species, such as antibodies having murineheavy and light chain variable regions linked to human constant regions.

The term “CDR-grafted antibody” refers to antibodies which compriseheavy and light chain variable region sequences from one species but inwhich the sequences of one or more of the CDR regions of VH and/or VLare replaced with CDR sequences of another species, such as antibodieshaving murine heavy and light chain variable regions in which one ormore of the murine CDRs (e.g., CDR3) has been replaced with human CDRsequences.

The term “humanized antibody” refers to antibodies which comprise heavyand light chain variable region sequences from a non-human species(e.g., a mouse) but in which at least a portion of the VH and/or VLsequence has been altered to be more “human-like”, i.e., more similar tohuman germline variable sequences. One type of humanized antibody is aCDR-grafted antibody, in which human CDR sequences are introduced intonon-human VH and VL sequences to replace the corresponding nonhuman CDRsequences.

Various aspects of the invention are described in further detail in thefollowing subsections.

II. Methods of Invention

Generally, diafiltration is a technique that uses membranes to remove,replace, or lower the concentration of salts or solvents from solutionscontaining proteins, peptides, nucleic acids, and other biomolecules.Protein production operations often involve final diafiltration of aprotein solution into a formulation buffer once the protein has beenpurified from impurities resulting from its expression, e.g., host cellproteins. The invention described herein provides a means for obtainingan aqueous formulation by subjecting a protein solution to diafiltrationusing water alone as a diafiltration solution. Thus, the formulation ofthe invention is based on using water as a formulation medium during thediafiltration process and does not rely on traditional formulationmediums which include excipients, such as surfactants, used tosolubilize and/or stabilize the protein in the final formulation. Theinvention provides a method for transferring a protein into pure waterfor use in a stable formulation, wherein the protein remains in solutionand is able to be concentrated at high levels without the use of otheragents to maintain its stability.

Prior to diafiltration or DF/UF in accordance with the teachings herein,the method includes first providing a protein in a first solution. Theprotein may be formulated in any first solution, including formulationsusing techniques that are well established in the art, such as synthetictechniques (e.g., recombinant techniques, peptide synthesis, or acombination thereof). Alternatively, the protein used in the methods andcompositions of the invention is isolated from an endogenous source ofthe protein. The initial protein solution may be obtained using apurification process whereby the protein is purified from aheterogeneous mix of proteins. In one embodiment, the initial proteinsolution used in the invention is obtained from a purification methodwhereby proteins, including antibodies, expressed in a mammalianexpression system are subjected to numerous chromatography steps whichremove host cell proteins (HCPs) from the protein solution. In oneembodiment, the first protein solution is obtained from a mammalian cellexpression system and has been purified to remove host cell proteins(HCPs). Examples of methods of purification are described in U.S.application Ser. No. 11/732,918 (US 20070292442), incorporated byreference herein. It should be noted that there is no specialpreparation of the first protein solution required in accordance withthe methods of the invention.

Proteins which may be used in the compositions and methods of theinvention may be any size, i.e., molecular weight (M_(w)). For example,the protein may have a M_(w) equal to or greater than about 1 kDa, aM_(w) equal to or greater than about 10 kDa, a M_(w) equal to or greaterthan about 47 kDa, a M_(w) equal to or greater than about 57 kDa, aM_(w) equal to or greater than about 100 kDa, a M_(w) equal to orgreater than about 150 kDa, a M_(w) equal to or greater than about 200kDa, or a M_(w) equal to or greater than about 250 kDa. Numbersintermediate to the above recited M_(w), e.g., 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104,105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118,119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132,133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146,147, 148, 149, 150, 151, 153, 153, 154, 155, 156, 157, 158, 159, 160,161, 162, 163, 164, 165, 166, 167, 168, 169, 170, and so forth, as wellas all other numbers recited herein, are also intended to be part ofthis invention. Ranges of values using a combination of any of the aboverecited values as upper and/or lower limits are intended to be includedin the scope of the invention. For example, proteins used in theinvention may range in size from 57 kDa to 250 kDa, from 56 kDa to 242kDa, from 60 kDa to 270 kDa, and so forth.

The methods of the invention also include diafiltration of a firstprotein solution that comprises at least two distinct proteins. Forexample, the protein solution may contain two or more types ofantibodies directed to different molecules or different epitopes of thesame molecule.

In one embodiment, the protein that is in solution is a therapeuticprotein, including, but not limited to, fusion proteins and enzymes.Examples of therapeutic proteins include, but are not limited to,Pulmozyme (Dornase alfa), Regranex (Becaplermin), Activase (Alteplase),Aldurazyme (Laronidase), Amevive (Alefacept), Aranesp (Darbepoetinalfa), Becaplermin Concentrate, Betaseron (Interferon beta-1b), BOTOX(Botulinum Toxin Type A), Elitek (Rasburicase), Elspar (Asparaginase),Epogen (Epoetin alfa), Enbrel (Etanercept), Fabrazyme (Agalsidase beta),Infergen (Interferon alfacon-1), Intron A (Interferon alfa-2a), Kineret(Anakinra), MYOBLOC (Botulinum Toxin Type B), Neulasta (Pegfilgrastim),Neumega (Oprelvekin), Neupogen (Filgrastim), Ontak (Denileukindiftitox), PEGASYS (Peginterferon alfa-2a), Proleukin (Aldesleukin),Pulmozyme (Dornase alfa), Rebif (Interferon beta-1a), Regranex(Becaplermin), Retavase (Reteplase), Roferon-A (Interferon alfa-2),TNKase (Tenecteplase), and Xigris (Drotrecogin alfa), Arcalyst(Rilonacept), NPlate (Romiplostim), Mircera (methoxypolyethyleneglycol-epoetin beta), Cinryze (C1 esterase inhibitor), Elaprase(idursulfase), Myozyme (alglucosidase alfa), Orencia (abatacept),Naglazyme (galsulfase), Kepivance (palifermin) and Actimmune (interferongamma-1b).

The protein used in the invention may also be an antibody, orantigen-binding fragment thereof. Examples of antibodies that may beused in the invention include chimeric antibodies, non-human antibodies,human antibodies, humanized antibodies, and domain antibodies (dAbs). Inone embodiment, the antibody, or antigen-binding fragment thereof, is ananti-TNFα and/or an anti-IL-12 antibody (e.g., it may be a dual variabledomain (DVD) antibody). Other examples of antibodies, or antigen-bindingfragments thereof, which may be used in the methods and compositions ofthe invention include, but are not limited to, 1D4.7 (anti-IL-12/IL-23antibody; Abbott Laboratories), 2.5(E)mg1 (anti-IL-18; AbbottLaboratories), 13C5.5 (anti-IL-13 antibody; Abbott Laboratories), J695(anti-IL-12; Abbott Laboratories), Afelimomab (Fab 2 anti-TNF; AbbottLaboratories), Humira (adalimumab) Abbott Laboratories), Campath(Alemtuzumab), CEA-Scan Arcitumomab (fab fragment), Erbitux (Cetuximab),Herceptin (Trastuzumab), Myoscint (Imciromab Pentetate), ProstaScint(Capromab Pendetide), Remicade (Infliximab), ReoPro (Abciximab), Rituxan(Rituximab), Simulect (Basiliximab), Synagis (Palivizumab), Verluma(Nofetumomab), Xolair (Omalizumab), Zenapax (Daclizumab), Zevalin(Ibritumomab Tiuxetan), Orthoclone OKT3 (Muromonab-CD3), Panorex(Edrecolomab), Mylotarg (Gemtuzumab ozogamicin), golimumab (Centocor),Cimzia (Certolizumab pegol), Soliris (Eculizumab), CNTO 1275(ustekinumab), Vectibix (panitumumab), Bexxar (tositumomab and I¹³¹tositumomab), an anti-IL-17 antibody Antibody 7 as described inInternational Application WO 2007/149032 (Cambridge AntibodyTechnology), the entire contents of which are incorporated by referenceherein, the anti-IL-13 antibody CAT-354 (Cambridge Antibody Technology),the anti-human CD4 antibody CE9y4PE (IDEC-151, clenoliximab) (BiogenIDEC/Glaxo Smith Kline), the anti-human CD4 antibody IDECCE9.1/SB-210396 (keliximab) (Biogen IDEC), the anti-human CD80 antibodyIDEC-114 (galiximab) (Biogen IDEC), the anti-Rabies Virus Proteinantibody CR4098 (foravirumab), and the anti-human TNF-relatedapoptosis-inducing ligand receptor 2 (TRAIL-2) antibody HGS-ETR2(lexatumumab) (Human Genome Sciences, Inc.), and Avastin (bevacizumab).

Techniques for the production of antibodies are provided below.

Polyclonal Antibodies

Polyclonal antibodies generally refer to a mixture of antibodies thatare specific to a certain antigen, but bind to different epitopes onsaid antigen. Polyclonal antibodies are generally raised in animals bymultiple subcutaneous (sc) or intraperitoneal (ip) injections of therelevant antigen and an adjuvant. It may be useful to conjugate therelevant antigen to a protein that is immunogenic in the species to beimmunized, e.g., keyhole limpet hemocyanin, serum albumin, bovinethyroglobulin, or soybean trypsin inhibitor using a bifunctional orderivatizing agent, for example, maleimidobenzoyl sulfosuccinimide ester(conjugation through cysteine residues), N-hydroxysuccinimide (throughlysine residues), glutaraldehyde, succinic anhydride, SOCl₂, or R₁NCNR,where R and R₁ are different alkyl groups. Methods for making polyclonalantibodies are known in the art, and are described, for example, inAntibodies: A Laboratory Manual, Lane and Harlow (1988), incorporated byreference herein.

Monoclonal Antibodies

A “monoclonal antibody” as used herein is intended to refer to ahybridoma-derived antibody (e.g., an antibody secreted by a hybridomaprepared by hybridoma technology, such as the standard Kohler andMilstein hybridoma methodology). For example, the monoclonal antibodiesmay be made using the hybridoma method first described by Kohler et al.,Nature, 256:495 (1975), or may be made by recombinant DNA methods (U.S.Pat. No. 4,816,567). Thus, a hybridoma-derived dual-specificity antibodyof the invention is still referred to as a monoclonal antibody althoughit has antigenic specificity for more than a single antigen.

Monoclonal antibodies are obtained from a population of substantiallyhomogeneous antibodies, i.e., the individual antibodies comprising thepopulation are identical except for possible naturally occurringmutations that may be present in minor amounts. Thus, the modifier“monoclonal” indicates the character of the antibody as not being amixture of discrete antibodies.

In a further embodiment, antibodies can be isolated from antibody phagelibraries generated using the techniques described in McCafferty et al.,Nature, 348:552-554 (1990). Clackson et al., Nature, 352:624-628 (1991)and Marks et al., J. Mol. Biol., 222:581-597 (1991) describe theisolation of murine and human antibodies, respectively, using phagelibraries. Subsequent publications describe the production of highaffinity (nM range) human antibodies by chain shuffling (Marks et al.,Bio/Technology, 10:779-783 (1992)), as well as combinatorial infectionand in vivo recombination as a strategy for constructing very largephage libraries (Waterhouse et al., Nuc. Acids. Res., 21:2265-2266(1993)). Thus, these techniques are viable alternatives to traditionalmonoclonal antibody hybridoma techniques for isolation of monoclonalantibodies.

Antibodies and antibody fragments may also be isolated from yeast andother eukaryotic cells with the use of expression libraries, asdescribed in U.S. Pat. Nos. 6,423,538; 6,696,251; 6,699,658; 6,300,065;6,399,763; and 6,114,147. Eukaryotic cells may be engineered to expresslibrary proteins, including from combinatorial antibody libraries, fordisplay on the cell surface, allowing for selection of particular cellscontaining library clones for antibodies with affinity to select targetmolecules. After recovery from an isolated cell, the library clonecoding for the antibody of interest can be expressed at high levels froma suitable mammalian cell line.

Additional methods for developing antibodies of interest includecell-free screening using nucleic acid display technology, as describedin U.S. Pat. Nos. 7,195,880; 6,951,725; 7,078,197; 7,022,479, 6,518,018;7,125,669; 6,846,655; 6,281,344; 6,207,446; 6,214,553; 6,258,558;6,261,804; 6,429,300; 6,489,116; 6,436,665; 6,537,749; 6,602,685;6,623,926; 6,416,950; 6,660,473; 6,312,927; 5,922,545; and 6,348,315.These methods can be used to transcribe a protein in vitro from anucleic acid in such a way that the protein is physically associated orbound to the nucleic acid from which it originated. By selecting for anexpressed protein with a target molecule, the nucleic acid that codesfor the protein is also selected. In one variation on cell-freescreening techniques, antibody sequences isolated from immune systemcells can be isolated and partially randomized polymerase chain reactionmutagenesis techniques to increase antibody diversity. These partiallyrandomized antibody genes are then expressed in a cell-free system, withconcurrent physical association created between the nucleic acid andantibody.

The DNA also may be modified, for example, by substituting the codingsequence for human heavy- and light-chain constant domains in place ofthe homologous murine sequences (U.S. Pat. No. 4,816,567; Morrison, etal., Proc. Natl. Acad. Sci. USA, 81:6851 (1984)), or by covalentlyjoining to the immunoglobulin coding sequence all or part of the codingsequence for a non-immunoglobulin polypeptide.

Typically such non-immunoglobulin polypeptides are substituted for theconstant domains of an antibody, or they are substituted for thevariable domains of one antigen-combining site of an antibody to createa chimeric bivalent antibody comprising one antigen-combining sitehaving specificity for an antigen and another antigen-combining sitehaving specificity for a different antigen.

Chimeric or hybrid antibodies also may be prepared in vitro using knownmethods in synthetic protein chemistry, including those involvingcrosslinking agents. For example, immunotoxins may be constructed usinga disulfide-exchange reaction or by forming a thioether bond. Examplesof suitable reagents for this purpose include iminothiolate andmethyl-4-mercaptobutyrimidate.

Humanized Antibodies

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and co-workers(Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature,332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), bysubstituting non-human (e.g., rodent) CDRs or CDR sequences for thecorresponding sequences of a human antibody. Accordingly, such“humanized” antibodies are chimeric antibodies (U.S. Pat. No.4,816,567), wherein substantially less than an intact human variabledomain has been substituted by the corresponding sequence from anon-human species. In practice, humanized antibodies are typically humanantibodies in which some CDR residues and possibly some framework (FR)residues are substituted by residues from analogous sites in rodentantibodies. Additional references which describe the humanizationprocess include Sims et al., J. Immunol., 151:2296 (1993); Chothia etal., J. Mol. Biol., 196:901 (1987); Carter et al., Proc. Natl. Acad.Sci. USA, 89:4285 (1992); Presta et al., J. Immunol., 151:2623 (1993),each of which is incorporated by reference herein.

Human Antibodies

Alternatively, it is now possible to produce transgenic animals (e.g.,mice) that are capable, upon immunization, of producing a fullrepertoire of human antibodies in the absence of endogenousimmunoglobulin production. For example, it has been described that thehomozygous deletion of the antibody heavy-chain joining region (JH) genein chimeric and germ-line mutant mice results in complete inhibition ofendogenous antibody production. Transfer of the human germ-lineimmunoglobulin gene array in such germ-line mutant mice will result inthe production of human antibodies upon antigen challenge. See, e.g.,Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 (1993);Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann et al., Yearin Immuno., 7:33 (1993). Human antibodies can also be derived fromphage-display libraries (Hoogenboom et al., J. Mol. Biol., 227:381(1991); Marks et al., J. Mol. Biol., 222:581-597 (1991)).

In one embodiment, the formulation of the invention comprises anantibody, or antigen-binding portion thereof, which binds human TNFα,including, for example, adalimumab (also referred to as Humira,adalimumab, or D2E7; Abbott Laboratories). In one embodiment, theantibody, or antigen-binding fragment thereof, dissociates from humanTNFα with a K_(d) of 1×10⁻⁸ M or less and a K_(off) rate constant of1×10⁻³ s⁻¹ or less, both determined by surface plasmon resonance, andneutralizes human TNFα cytotoxicity in a standard in vitro L929 assaywith an IC₅₀ of 1×10⁻⁷ M or less. Examples and methods for making human,neutralizing antibodies which have a high affinity for human TNFα,including sequences of the antibodies, are described in U.S. Pat. No.6,090,382 (referred to as D2E7), incorporated by reference herein.

In one embodiment, the human neutralizing, antibody, or anantigen-binding portion thereof, having a high affinity for human TNFαis an isolated human antibody, or an antigen-binding portion thereof,with a light chain variable region (LCVR) having a CDR3 domaincomprising the amino acid sequence of SEQ ID NO:3, or modified from SEQID NO:3 by a single alanine substitution at position 1, 4, 5, 7 or 8,and with a heavy chain variable region (HCVR) having a CDR3 domaincomprising the amino acid sequence of SEQ ID NO:4, or modified from SEQID NO:4 by a single alanine substitution at position 2, 3, 4, 5, 6, 8,9, 10 or 11. Preferably, the LCVR further has a CDR2 domain comprisingthe amino acid sequence of SEQ ID NO:5 (i.e., the D2E7 VL CDR2) and theHCVR further has a CDR2 domain comprising the amino acid sequence of SEQID NO: 6 (i.e., the D2E7 VH CDR2). Even more preferably, the LCVRfurther has CDR1 domain comprising the amino acid sequence of SEQ IDNO:7 (i.e., the D2E7 VL CDR1) and the HCVR has a CDR1 domain comprisingthe amino acid sequence of SEQ ID NO:8 (i.e., the D2E7 VH CDR1).

In another embodiment, the human neutralizing, antibody, or anantigen-binding portion thereof, having a high affinity for human TNFαis an isolated human antibody, or an antigen-binding portion thereof,with a light chain variable region (LCVR) comprising the amino acidsequence of SEQ ID NO:1 (i.e., the D2E7 VL) and a heavy chain variableregion (HCVR) comprising the amino acid sequence of SEQ ID NO:2 (i.e.,the D2E7 VH). In certain embodiments, the antibody comprises a heavychain constant region, such as an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgMor IgD constant region. Preferably, the heavy chain constant region isan IgG1 heavy chain constant region or an IgG4 heavy chain constantregion. Furthermore, the antibody can comprise a light chain constantregion, either a kappa light chain constant region or a lambda lightchain constant region. Preferably, the antibody comprises a kappa lightchain constant region.

In one embodiment, the formulation of the invention comprises anantibody, or antigen-binding portion thereof, which binds human IL-12,including, for example, the antibody J695 (Abbott Laboratories; alsoreferred to as ABT-874) (U.S. Pat. No. 6,914,128). J695 is a fully humanmonoclonal antibody designed to target and neutralize interleukin-12 andinterleukin-23. In one embodiment, the antibody, or antigen-bindingfragment thereof, has the following characteristics: it dissociates fromhuman IL-1α with a K_(D) of 3×10⁻⁷ M or less; dissociates from humanIL-1β with a K_(D) of 5×10⁻⁵ M or less; and does not bind mouse IL-1α ormouse IL-1β. Examples and methods for making human, neutralizingantibodies which have a high affinity for human IL-12, includingsequences of the antibody, are described in U.S. Pat. No. 6,914,128,incorporated by reference herein.

In one embodiment, the formulation of the invention comprises anantibody, or antigen-binding portion thereof, which binds human IL-18,including, for example, the antibody 2.5(E)mg1 (Abbott Bioresearch; alsoreferred to as ABT-325) (see U.S. Patent Application No. 2005/0147610,incorporated by reference herein).

In one embodiment, the formulation of the invention comprises ananti-IL-12/anti-IL-23 antibody, or antigen-binding portion thereof,which is the antibody 1D4.7 (Abbott Laboratories; also referred to asABT-147) (see WO 2007/005608 A2, published Jan. 11, 2007, incorporatedby reference herein).

In one embodiment, the formulation of the invention comprises ananti-IL-13 antibody, or antigen-binding portion thereof, which is theantibody 13C5.5 (Abbott Laboratories; also referred to as ABT-308) (see.PCT/US2007/19660 (WO 08/127271), incorporated by reference herein).

In one embodiment, the formulation of the invention comprises anantibody, or antigen-binding portion thereof, which is the antibody 7C6,an anti-amyloid β antibody (Abbott Laboratories; see PCT publication WO07/062852, incorporated by reference herein).

Bispecific Antibodies

Bispecific antibodies (BsAbs) are antibodies that have bindingspecificities for at least two different epitopes. Such antibodies canbe derived from full length antibodies or antibody fragments (e.g.,F(ab′)2 bispecific antibodies).

Methods for making bispecific antibodies are known in the art.Traditional production of full length bispecific antibodies is based onthe coexpression of two immunoglobulin heavy chain-light chain pairs,where the two chains have different specificities (Millstein et al.,Nature, 305:537-539 (1983)). Because of the random assortment ofimmunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of 10 different antibody molecules, of whichonly one has the correct bispecific structure. Purification of thecorrect molecule, which is usually done by affinity chromatographysteps, is rather cumbersome, and the product yields are low. Similarprocedures are disclosed in WO 93/08829 and in Traunecker et al., EMBOJ., 10:3655-3659 (1991).

According to a different approach, antibody variable domains with thedesired binding specificities (antibody-antigen combining sites) arefused to immunoglobulin constant domain sequences.

The fusion preferably is with an immunoglobulin heavy chain constantdomain, comprising at least part of the hinge, CH2, and CH3 regions. Itis preferred to have the first heavy-chain constant region (CH1)containing the site necessary for light chain binding, present in atleast one of the fusions. DNAs encoding the immunoglobulin heavy chainfusions and, if desired, the immunoglobulin light chain, are insertedinto separate expression vectors, and are co-transfected into a suitablehost organism. This provides for great flexibility in adjusting themutual proportions of the three polypeptide fragments in embodimentswhen unequal ratios of the three polypeptide chains used in theconstruction provide the optimum yields. It is, however, possible toinsert the coding sequences for two or all three polypeptide chains inone expression vector when the expression of at least two polypeptidechains in equal ratios results in high yields or when the ratios are ofno particular significance.

In a preferred embodiment of this approach, the bispecific antibodiesare composed of a hybrid immunoglobulin heavy chain with a first bindingspecificity in one arm, and a hybrid immunoglobulin heavy chain-lightchain pair (providing a second binding specificity) in the other arm. Itwas found that this asymmetric structure facilitates the separation ofthe desired bispecific compound from unwanted immunoglobulin chaincombinations, as the presence of an immunoglobulin light chain in onlyone half of the bispecific molecule provides for a facile way ofseparation. This approach is disclosed in WO 94/04690 published Mar. 3,1994. For further details of generating bispecific antibodies see, forexample, Suresh et al., Methods in Enzymology, 121:210 (1986).

Bispecific antibodies include cross-linked or “heteroconjugate”antibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin, the other to biotin. Such antibodies have, forexample, been proposed to target immune system cells to unwanted cells(U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO91/00360, WO 92/200373, and EP 03089). Heteroconjugate antibodies may bemade using any convenient cross-linking methods. Suitable cross-linkingagents are well known in the art, and are disclosed in U.S. Pat. No.4,676,980, along with a number of cross-linking techniques.

Techniques for generating bispecific antibodies from antibody fragmentshave also been described in the literature. The following techniques canalso be used for the production of bivalent antibody fragments which arenot necessarily bispecific. For example, Fab′ fragments recovered fromE. coli can be chemically coupled in vitro to form bivalent antibodies.See, Shalaby et al., J. Exp. Med., 175:217-225 (1992).

Various techniques for making and isolating bivalent antibody fragmentsdirectly from recombinant cell culture have also been described. Forexample, bivalent heterodimers have been produced using leucine zippers.Kostelny et al., J. Immunol., 148(5): 1547-1553 (1992). The leucinezipper peptides from the Fos and Jun proteins were linked to the Fab′portions of two different antibodies by gene fusion. The antibodyhomodimers were reduced at the hinge region to form monomers and thenre-oxidized to form the antibody heterodimers. The “diabody” technologydescribed by Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448(1993) has provided an alternative mechanism for makingbispecific/bivalent antibody fragments. The fragments comprise aheavy-chain variable domain (VH) connected to a light-chain variabledomain (VL) by a linker which is too short to allow pairing between thetwo domains on the same chain. Accordingly, the VH and VL domains of onefragment are forced to pair with the complementary VL and VH domains ofanother fragment, thereby forming two antigen-binding sites. Anotherstrategy for making bispecific/bivalent antibody fragments by the use ofsingle-chain Fv (sFv) dimers has also been reported. See Gruber et al.,J. Immunol., 152:5368 (1994).

In one embodiment, the formulation of the invention comprises anantibody which is bispecific for IL-1 (including IL-1α and IL-1β).Examples and methods for making bispecific IL-1 antibodies can be foundin U.S. Provisional Appln. No. 60/878,165, filed Dec. 29, 2006.

Diafiltration/Ultrafiltration (also generally referred to herein asDF/UF) selectively utilizes permeable (porous) membrane filters toseparate the components of solutions and suspensions based on theirmolecular size. A membrane retains molecules that are larger than thepores of the membrane while smaller molecules such as salts, solventsand water, which are permeable, freely pass through the membrane. Thesolution retained by the membrane is known as the concentrate orretentate. The solution that passes through the membrane is known as thefiltrate or permeate. One parameter for selecting a membrane forconcentration is its retention characteristics for the sample to beconcentrated. As a general rule, the molecular weight cut-off (MWCO) ofthe membrane should be ⅓rd to ⅙th the molecular weight of the moleculeto be retained. This is to assure complete retention. The closer theMWCO is to that of the sample, the greater the risk for some smallproduct loss during concentration. Examples of membranes that can beused with methods of the invention include Omega™ PES membrane (30 kDaMWCO, i.e. molecules larger than 30 kDa are retained by the membrane andmolecules less than 30 kDa are allowed to pass to the filtrate side ofthe membrane) (Pall Corp., Port Washington, N.Y.); Millex®-GV SyringeDriven Filter Unit, PVDF 0.22 μm (Millipore Corp., Billerica, Mass.);Millex®-GP Syringe Driven Filter Unit, PES 0.22 μm; Sterivex®0.22 μmFilter Unit (Millipore Corp., Billerica, Mass.); and Vivaspinconcentrators (MWCO 10 kDa, PES; MWCO 3 kDa, PES) (Sartorius Corp.,Edgewood, N.Y.). In order to prepare a low-ionic protein formulation ofthe invention, the protein solution (which may be solubilized in abuffered formulation) is subjected to a DF/UF process, whereby water isused as a DF/UF medium. In a preferred embodiment, the DF/UF mediumconsists of water and does not include any other excipients.

Any water can be used in the DF/UF process of the invention, although apreferred water is purified or deionized water. Types of water known inthe art that may be used in the practice of the invention include waterfor injection (WFI) (e.g., HyPure WFI Quality Water (HyClone),AQUA-NOVA® WFI (Aqua Nova)), UltraPure™ Water (Invitrogen), anddistilled water (Invitrogen; Sigma-Aldrich).

There are two forms of DF/UF, including DF/UF in discontinuous mode andDF/UF in continuous mode. The methods of the invention may be performedaccording to either mode.

Continuous DF/UF (also referred to as constant volume DF/UF) involveswashing out the original buffer salts (or other low molecular weightspecies) in the retentate (sample or first protein solution) by addingwater or a new buffer to the retentate at the same rate as filtrate isbeing generated. As a result, the retentate volume and productconcentration does not change during the DF/UF process. The amount ofsalt removed is related to the filtrate volume generated, relative tothe retentate volume. The filtrate volume generated is usually referredto in terms of “diafiltration volumes”. A single diafiltration volume(DV) is the volume of retentate when diafiltration is started. Forcontinuous diafiltration, liquid is added at the same rate as filtrateis generated. When the volume of filtrate collected equals the startingretentate volume, 1 DV has been processed.

Discontinuous DF/UF (examples of which are provided below in theExamples section) includes two different methods, discontinuoussequential DF/UF and volume reduction discontinuous DF/UF. DiscontinuousDF/UF by sequential dilution involves first diluting the sample (orfirst protein solution) with water to a predetermined volume. Thediluted sample is then concentrated back to its original volume by UF.Discontinuous DF/UF by volume reduction involves first concentrating thesample to a predetermined volume, then diluting the sample back to itsoriginal volume with water or replacement buffer. As with continuousDF/UF, the process is repeated until the level of unwanted solutes,e.g., ionic excipients, are removed.

DF/UF may be performed in accordance with conventional techniques knownin the art using water, e.g, WFI, as the DF/UF medium (e.g., IndustrialUltrafiltration Design and Application of Diafiltration Processes,Beaton & Klinkowski, J. Separ. Proc. Technol., 4(2) 1-10 (1983)).Examples of commercially available equipment for performing DF/UFinclude Millipore Labscale™ TFF System (Millipore), LV Centramate™ LabTangential Flow System (Pall Corporation), and the UniFlux System (GEHealthcare).

For example, in a preferred embodiment, the Millipore Labscale™Tangential Flow Filtration (TFF) system with a 500 mL reservoir is usedto perform a method of the invention to produce a diafiltered antibodysolution. The DF/UF procedure is performed in a discontinuous manner,with 14 process steps used to produce a high concentration antibodyformulation in water. For additional exemplary equipment, solution andwater volumes, number of process steps, and other parameters ofparticular embodiments of the invention, see the Examples section below.

Alternative methods to diafiltration for buffer exchange where a proteinis re-formulated into water in accordance with the invention includedialysis and gel filtration, both of which are techniques known to thosein the art. Dialysis requires filling a dialysis bag (membrane casing ofdefined porosity), tying off the bag, and placing the bag in a bath ofwater. Through diffusion, the concentration of salt in the bag willequilibrate with that in the bath, wherein large molecules, e.g.,proteins that cannot diffuse through the bag remain in the bag. Thegreater the volume of the bath relative to the sample volume in thebags, the lower the equilibration concentration that can be reached.Generally, replacements of the bath water are required to completelyremove all of the salt. Gel filtration is a non-adsorptivechromatography technique that separates molecules on the basis ofmolecular size. In gel filtration, large molecules, e.g., proteins, maybe separated from smaller molecules, e.g., salts, by size exclusion.

In a preferred embodiment of the invention, the first protein solutionis subjected to a repeated volume exchange with the water, such that anaqueous formulation, which is essentially water and protein, isachieved. The diafiltration step may be performed any number of times,depending on the protein in solution, wherein one diafiltration stepequals one total volume exchange. In one embodiment, the diafiltrationprocess is performed 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to as many timesare deemed necessary to remove excipients, e.g., salts, from the firstprotein solution, such that the protein is dissolved essentially inwater. A single round or step of diafiltration is achieved when a volumeof water has been added to the retentate side that is equal to thestarting volume of the protein solution.

In one embodiment, the protein solution is subjected to at least 2diafiltration steps. In one embodiment, the diafiltration step or volumeexchange with water may be repeated at least four times, and preferablyat least five times. In one embodiment, the first protein solution issubjected to diafiltration with water until at least a six-fold volumeexchange is achieved. In another embodiment, the first protein solutionis subjected to diafiltration with water until at least a seven-foldvolume exchange is achieved. Ranges intermediate to the above recitednumbers, e.g., 4 to 6 or 5 to 7, are also intended to be part of thisinvention. For example, ranges of values using a combination of any ofthe above recited values as upper and/or lower limits are intended to beincluded.

In a preferred embodiment, loss of protein to the filtrate side of anultrafiltration membrane should be minimized. The risk of protein lossto the filtrate side of a particular membrane varies in relation to thesize of the protein relative to the membrane's pore size, and theprotein's concentration. With increases in protein concentration, riskof protein loss to the filtrate increases. For a particular membranepore size, risk of protein loss is greater for a smaller protein that isclose in size to the membrane's MWCO than it is for a larger protein.Thus, when performing DF/UF on a smaller protein, it may not be possibleto achieve the same reduction in volume, as compared to performing DF/UFon a larger protein using the same membrane, without incurringunacceptable protein losses. In other words, as compared to theultrafiltration of a solution of a smaller protein using the sameequipment and membrane, a solution of a larger protein could beultrafiltered to a smaller volume, with a concurrent higherconcentration of protein in the solution. DF/UF procedures using aparticular pore size membrane may require more process steps for asmaller protein than for a larger protein; a greater volume reductionand concentration for a larger protein permits larger volumes of waterto be added back, leading to a larger dilution of the remaining bufferor excipient ingredients in the protein solution for that individualprocess step. Fewer process steps may therefore be needed to achieve acertain reduction in solutes for a larger protein than for a smallerone. A person with skill in the art would be able to calculate theamount of concentration possible with each process step and the numberof overall process steps required to achieve a certain reduction insolutes, given the protein size and the pore size of the ultrafiltrationdevice to be used in the procedure.

As a result of the diafiltration methods of the invention, theconcentration of solutes in the first protein solution is significantlyreduced in the final aqueous formulation comprising essentially waterand protein. For example, the aqueous formulation may have a finalconcentration of excipients which is at least 95% less than the firstprotein solution, and preferably at least 99% less than the firstprotein solution. For example, in one embodiment, to dissolve a proteinin WFI is a process that creates a theoretical final excipientconcentration, reached by constant volume diafiltration with fivediafiltration volumes, that is equal or approximate to Ci e⁻⁵=0.00674,i.e., an approximate 99.3% maximum excipient reduction. In oneembodiment, a person with skill in the art may perform 6 volumeexchanges during the last step of a commercial DF/UF with constantvolume diafiltration, i.e., Ci would be C_(i) e⁻⁶=0.0025. This wouldprovide an approximate 99.75% maximum theoretical excipient reduction.In another embodiment, a person with skill in the art may use 8diafiltration volume exchanges to obtain a theoretical ˜99.9% maximumexcipient reduction.

The term “excipient-free” or “free of excipients” indicates that theformulation is essentially free of excipients. In one embodiment,excipient-free indicates buffer-free, salt-free, sugar-free, aminoacid-free, surfactant-free, and/or polyol free. In one embodiment, theterm “essentially free of excipients” indicates that the solution orformulation is at least 99% free of excipients. It should be noted,however, that in certain embodiments, a formulation may comprise acertain specified non-ionic excipient, e.g., sucrose or mannitol, andyet the formulation is otherwise excipient free. For example, aformulation may comprise water, a protein, and mannitol, wherein theformulation is otherwise excipient free. In another example, aformulation may comprise water, a protein, and polysorbate 80, whereinthe formulation is otherwise excipient free. In yet another example, theformulation may comprise water, a protein, a sorbitol, and polysorbate80, wherein the formulation is otherwise excipient free.

When water is used for diafiltering a first protein solution inaccordance with the methods described herein, ionic excipients will bewashed out, and, as a result, the conductivity of the diafilteredaqueous formulation is lower than the first protein solution. If anaqueous solution conducts electricity, then it must contain ions, asfound with ionic excipients. A low conductivity measurement is thereforeindicative that the aqueous formulation of the invention hassignificantly reduced excipients, including ionic excipients.

Conductivity of a solution is measured according to methods known in theart. Conductivity meters and cells may be used to determine theconductivity of the aqueous formulation, and should be calibrated to astandard solution before use. Examples of conductivity meters availablein the art include MYRON L Digital (Cole Parmer®), Conductometer(Metrohm AG), and Series 3105/3115 Integrated Conductivity Analyzers(Kemotron). In one embodiment, the aqueous formulation has aconductivity of less than 3 mS/cm. In another embodiment, the aqueousformulation has a conductivity of less than 2 mS/cm. In yet anotherembodiment, the aqueous formulation has a conductivity of less than 1mS/cm. In one aspect of the invention, the aqueous formulation has aconductivity of less than 0.5 mS/cm. Ranges intermediate to the aboverecited numbers, e.g., 1 to 3 mS/cm, are also intended to be encompassedby the invention. For example, ranges of values using a combination ofany of the above recited values as upper and/or lower limits areintended to be included. In addition, values that fall within therecited numbers are also included in the invention, e.g., 0.5, 0.6, 0.7,0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1,2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0 and so forth.

An important aspect of the invention is that the diafiltered proteinsolution (solution obtained following the diafiltration process of thefirst protein solution) can be concentrated. By following this process,it has been discovered that high concentrations of protein are stable inwater. Concentration following diafiltration results in an aqueousformulation containing water and an increased protein concentrationrelative to the first protein solution. Thus, the invention alsoincludes diafiltering a protein solution using water as a diafiltrationmedium and subsequently concentrating the resulting aqueous solution.Concentration of the diafiltered protein solution may be achievedthrough means known in the art, including centrifugation. For example,following diafiltration, the water-based diafiltrated protein solutionis subjected to a centrifugation process which serves to concentrate theprotein via ultrafiltration into a high concentration formulation whilemaintaining the water-based solution. Means for concentrating a solutionvia centrifugation with ultrafiltration membranes and/or devices areknown in the art, e.g., with Vivaspin centrifugal concentrators(Sartorius Corp. Edgewood, N.Y.).

The methods of the invention provide a means of concentrating a proteinat very high levels in water without the need for additional stabilizingagents. The concentration of the protein in the aqueous formulationobtained using the methods of the invention can be any amount inaccordance with the desired concentration. For example, theconcentration of protein in an aqueous solution made according to themethods herein is at least about 10 μg/mL; at least about 1 mg/mL; atleast about 10 mg/mL; at least about 20 mg/mL; at least about 50 mg/mL;at least about 75 mg/mL; at least about 100 mg/mL; at least about 125mg/mL; at least about 150 mg/mL; at least about 175 mg/mL; at leastabout 200 mg/mL; at least about 220 mg/mL; at least about 250 mg/mL; atleast about 300 mg/mL; or greater than about 300 mg/mL. Rangesintermediate to the above recited concentrations, e.g., at least about113 mg/mL, at least about 214 mg/mL, and at least about 300 mg/mL, arealso intended to be encompassed by the invention. In addition, ranges ofvalues using a combination of any of the above recited values (or valuesbetween the ranges described above) as upper and/or lower limits areintended to be included, e.g., 100 to 125 mg/mL, 113 to 125 mg/mL, and126 to 200 mg/mL or more.

The methods of the invention provide the advantage that the resultingformulation has a low percentage of protein aggregates, despite the highconcentration of the aqueous protein formulation. In one embodiment, theaqueous formulations comprising water and a high concentration of aprotein, e.g., antibodies, contains less than about 5% proteinaggregates, even in the absence of a surfactant or other type ofexcipient. In one embodiment, the formulation comprises no more thanabout 7.3% aggregate protein; the formulation comprises no more thanabout 5% aggregate protein; the formulation comprises no more than about4% aggregate protein; the formulation comprises no more than about 3%aggregate protein; the formulation comprises no more than about 2%aggregate protein; or the formulation comprising no more than about 1%aggregate protein. In one embodiment, the formulation comprises at leastabout 92%, at least about 93%, at least about 94%, at least about 95%,at least about 96%, at least about 97%, at least about 98%, or at leastabout 99% monomer protein. Ranges intermediate to the above recitedconcentrations, e.g., at least about 98.6%, no more than about 4.2%, arealso intended to be part of this invention. In addition, ranges ofvalues using a combination of any of the above recited values as upperand/or lower limits are intended to be included.

Many protein-based pharmaceutical products need to be formulated at highconcentrations. For example, antibody-based products increasingly tendto exceed 100 mg/mL in their Drug Product (DP) formulation to achieveappropriate efficacy and meet a typical patient usability requirement ofa maximal ˜1 mL injection volume. Accordingly, downstream processingsteps, such as diafiltration into the final formulation buffer orultrafiltration to increase the protein concentration, are alsoconducted at higher concentrations.

Classic thermodynamics predicts that intermolecular interactions canaffect the partitioning of small solutes across a dialysis membrane,especially at higher protein concentrations, and models describingnon-ideal dialysis equilibrium and the effects of intermolecularinteractions are available (Tanford Physical chemistry ormacromolecules. New York, John Wiley and Sons, Inc., p. 182, 1961;Tester and Modell Thermodynamics and its applications, 3^(rd) ed. UpperSaddle River, NL, Prentice-Hall, 1997). In the absence of theavailability of detailed thermodynamic data in the process developmentenvironment, which is necessary to apply these type of models,intermolecular interactions rarely are taken into account during thedesign of commercial DF/UF operations. Consequently, DP excipientconcentrations may differ significantly from the concentration labeled.Several examples of this discrepancy in commercial and developmentproducts are published, e.g., chloride being up to 30% lower thanlabeled in an IL-1 receptor antagonist, histidine being 40% lower thanlabeled in a PEG-sTNF receptor, and acetate being up to 200% higher thanlabeled in a fusion conjugate protein (Stoner et al., J. Pharm. Sci.,93, 2332-2342 (2004)). There are several reasons why the actual DP maybe different from the composition of the buffer the protein isdiafiltered into, including the Donnan effect (Tombs and Peacocke (1974)Oxford; Clarendon Press), non-specific interactions (Arakawa andTimasheff, Arch. Biochem. Biophys., 224, 169-77 (1983); Timasheff, Annu.Rev. Biophys. Biomol. Struct., 22, 67-97 (1993)), and volume exclusioneffects. Volume exclusion includes most protein partial specific volumesare between 0.7 and 0.8 mL/g.⁵ Thus, for a globular protein at 100mg/mL, protein molecules occupy approx. 7.5% of the total solutionvolume. No significant intermolecular interactions assumed, this wouldtranslate to a solute molar concentration on the retentate side of themembrane that is 92.5% of the molar concentration on the permeate sideof the membrane. This explains why basically all protein solutioncompositions necessarily change during ultrafiltration processing. Forinstance, at 40 mg/mL the protein molecules occupy approx. 3% of thetotal solution volume, and an ultrafiltration step increasing theconcentration to 150 mg/mL will necessarily induce molar excipientconcentrations to change by more than 8% (as protein at 150 mg/mLaccounts for more than 11% of total solution volume). Rangesintermediate to the above recited percentages are also intended to bepart of this invention. In addition, ranges of values using acombination of any of the above recited values as upper and/or lowerlimits are intended to be included.

In accordance with the methods and compositions of the invention, buffercomposition changes during DF/UF operations can be circumvented by usingpure water as diafiltration medium. By concentrating the protein ˜20%more than the concentration desired in the final Bulk DS, excipientscould subsequently be added, for instance, via highly concentratedexcipient stock solutions. Excipient concentrations and solution pHcould then be guaranteed to be identical as labeled.

The aqueous formulation of the invention provides an advantage as astarting material, as it essentially contains no excipient. Anyexcipient(s) which is added to the formulation following thediafiltration in water can be accurately calculated, i.e., pre-existingconcentrations of excipient(s) do not interfere with the calculation.Examples of pharmaceutically acceptable excipients are described inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980),incorporated by reference herein. Thus, another aspect of the inventionincludes using the aqueous formulation obtained through the methodsdescribed herein, for the preparation of a formulation, particularly apharmaceutical formulation, having known concentrations of excipient(s),including non-ionic excipient(s) or ionic excipient(s). One aspect ofthe invention includes an additional step where an excipient(s) is addedto the aqueous formulation comprising water and protein. Thus, themethods of the invention provide an aqueous formulation which isessentially free of excipients and may be used as a starting materialfor preparing formulations comprising water, proteins, and specificconcentrations of excipients.

In one embodiment, the methods of the invention may be used to addnon-ionic excipients, e.g., sugars or non-ionic surfactants, such aspolysorbates and poloxamers, to the formulation without changing thecharacteristics, e.g., protein concentration, hydrodynamic diameter ofthe protein, conductivity, etc.

Additional characteristics and advantages of aqueous formulationsobtained using the above methods are described below in section III.Exemplary protocols for performing the methods of the invention are alsodescribed below in the Examples.

III. Formulations of Invention

The invention provide an aqueous formulation comprising a protein andwater which has a number of advantages over conventional formulations inthe art, including stability of the protein in water without therequirement for additional excipients, increased concentrations ofprotein without the need for additional excipients to maintainsolubility of the protein, and low osmolality. The formulations of theinvention also have advantageous storage properties, as the proteins inthe formulation remain stable during storage, e.g., stored as a liquidform for more than 3 months at 7° C. or freeze/thaw conditions, even athigh protein concentrations and repeated freeze/thaw processing steps.In one embodiment, formulations of the invention include highconcentrations of proteins such that the aqueous formulation does notshow significant opalescence, aggregation, or precipitation.

The aqueous formulation of the invention does not rely on standardexcipients, e.g., a tonicity modifier, a stabilizing agent, asurfactant, an anti-oxidant, a cryoprotectant, a bulking agent, alyroprotectant, a basic component, and an acidic component. In otherembodiments of the invention, the formulation contains water, one ormore proteins, and no ionic excipients (e.g., salts, free amino acids).

In certain embodiments, the aqueous formulation of the inventioncomprises a protein concentration of at least 50 mg/mL and water,wherein the formulation has an osmolality of no more than 30 mOsmol/kg.Lower limits of osmolality of the aqueous formulation are alsoencompassed by the invention. In one embodiment the osmolality of theaqueous formulation is no more than 15 mOsmol/kg. The aqueousformulation of the invention may have an osmolality of less than 30mOsmol/kg, and also have a high protein concentration, e.g., theconcentration of the protein is at least 100 mg/mL, and may be as muchas 200 mg/mL or greater. Ranges intermediate to the above recitedconcentrations and osmolality units are also intended to be part of thisinvention. In addition, ranges of values using a combination of any ofthe above recited values as upper and/or lower limits are intended to beincluded.

The concentration of the aqueous formulation of the invention is notlimited by the protein size and the formulation may include any sizerange of proteins. Included within the scope of the invention is anaqueous formulation comprising at least 50 mg/mL and as much as 200mg/mL or more of a protein, which may range in size from 5 kDa to 150kDa or more. In one embodiment, the protein in the formulation of theinvention is at least about 15 kD in size, at least about 20 kD in size;at least about 47 kD in size; at least about 60 kD in size; at leastabout 80 kD in size; at least about 100 kD in size; at least about 120kD in size; at least about 140 kD in size; at least about 160 kD insize; or greater than about 160 kD in size. Ranges intermediate to theabove recited sizes are also intended to be part of this invention. Inaddition, ranges of values using a combination of any of the aboverecited values as upper and/or lower limits are intended to be included.

The aqueous formulation of the invention may be characterized by thehydrodynamic diameter (D_(h)) of the proteins in solution. Thehydrodynamic diameter of the protein in solution may be measured usingdynamic light scattering (DLS), which is an established analyticalmethod for determining the D_(h) of proteins. Typical values formonoclonal antibodies, e.g., IgG, are about 10 nm. Low-ionicformulations, like those described herein, may be characterized in thatthe D_(h) of the proteins are notably lower than protein formulationscomprising ionic excipients. It has been discovered that the D_(h)values of antibodies in aqueous formulations made using the DF/UFprocess using pure water as an exchange medium, are notably lower thanthe D_(h) of antibodies in conventional formulations independent ofprotein concentration. In one embodiment, antibodies in the aqueousformulation of the invention have a D_(h) of less than 4 nm, or lessthan 3 nm.

In one embodiment, the D_(h) of the protein in the aqueous formulationis smaller relative to the D_(h) of the same protein in a bufferedsolution, irrespective of protein concentration. Thus, in certainembodiments, protein in an aqueous formulation made in accordance withthe methods described herein, will have a D_(h) which is at least 25%less than the D_(h) of the protein in a buffered solution at the samegiven concentration. Examples of buffered solutions include, but are notlimited to phosphate buffered saline (PBS). In certain embodiments,proteins in the aqueous formulation of the invention have a D_(h) thatis at least 50% less than the D_(h) of the protein in PBS in at thegiven concentration; at least 60% less than the D_(h) of the protein inPBS at the given concentration; at least 70% less than the D_(h) of theprotein in PBS at the given concentration; or more than 70% less thanthe D_(h) of the protein in PBS at the given concentration. Rangesintermediate to the above recited percentages are also intended to bepart of this invention, e.g., 55%, 56%, 57%, 64%, 68%, and so forth. Inaddition, ranges of values using a combination of any of the aboverecited values as upper and/or lower limits are intended to be included,e.g., 50% to 80%.

Protein aggregation is a common problem in protein solutions, and oftenresults from increased concentration of the protein. The instantinvention provides a means for achieving a high concentration, lowprotein aggregation formulation. Formulations of the invention do notrely on a buffering system and excipients, including surfactants, tokeep proteins in the formulation soluble and from aggregating.Formulations of the invention can be advantageous for therapeuticpurposes, as they are high in protein concentration and water-based, notrelying on other agents to achieve high, stable concentrations ofproteins in solution.

The majority of biologic products (including antibodies) are subject tonumerous degradative processes which frequently arise from non-enzymaticreactions in solution. These reactions may have a long-term impact onproduct stability, safety and efficacy. These instabilities can beretarded, if not eliminated, by storage of product at subzerotemperatures, thus gaining a tremendous advantage for the manufacturerin terms of flexibility and availability of supplies over the productlife-cycle. Although freezing is often the safest and most reliablemethod of biologics product storage, it has inherent risks. Freezing caninduce stress in proteins through cold denaturation, by introducingice-liquid interfaces, and by freeze-concentration (cryoconcentration)of solutes when the water crystallizes.

Cryoconcentration is a process in which a flat, uncontrolled moving icefront is formed during freezing that excludes solute molecules (smallmolecules such as sucrose, salts, and other excipients typically used inprotein formulation, or macromolecules such as proteins), leading tozones in which proteins may be found at relatively high concentration inthe presence of other solutes at concentrations which may potentiallylead to local pH or ionic concentration extremes. For most proteins,these conditions can lead to denaturation and in some cases, protein andsolute precipitation. Since buffer salts and other solutes are alsoconcentrated under such conditions, these components may reachconcentrations high enough to lead to pH and/or redox changes in zoneswithin the frozen mass. The pH shifts observed as a consequence ofbuffer salt crystallization (e.g., phosphates) in the solutions duringfreezing can span several pH units, which may impact protein stability.

Concentrated solutes may also lead to a depression of the freezing pointto an extent where the solutes may not be frozen at all, and proteinswill exist within a solution under these adverse conditions. Often,rapid cooling may be applied to reduce the time period the protein isexposed to these undesired conditions. However, rapid freezing caninduce a large-area ice-water interface, whereas slow cooling inducessmaller interface areas. For instance, rapid cooling of six modelproteins during one freeze/thaw step was shown to reveal a denaturationeffect greater than 10 cycles of slow cooling, demonstrating the greatdestabilization potential of hydrophobic ice surface-induceddenaturation.

The aqueous formulation of the invention has advantageous stability andstorage properties. Stability of the aqueous formulation is notdependent on the form of storage, and includes, but is not limited to,formulations which are frozen, lyophilized, or spray-dried. Stabilitycan be measured at a selected temperature for a selected time period. Inone aspect of the invention, the protein in the aqueous formulations isstable in a liquid form for at least 3 months; at least 4 months, atleast 5 months; at least 6 months; at least 12 months. Rangesintermediate to the above recited time periods are also intended to bepart of this invention, e.g., 9 months, and so forth. In addition,ranges of values using a combination of any of the above recited valuesas upper and/or lower limits are intended to be included. Preferably,the formulation is stable at room temperature (about 30° C.) or at 40°C. for at least 1 month and/or stable at about 2-8° C. for at least 1year, or more preferably stable at about 2-8° C. for at least 2 years.Furthermore, the formulation is preferably stable following freezing(to, e.g., −80° C.) and thawing of the formulation, hereinafter referredto as a “freeze/thaw cycle.”

Stability of a protein can be also be defined as the ability to remainbiologically active. A protein “retains its biological activity” in apharmaceutical formulation, if the protein in a pharmaceuticalformulation is biologically active upon administration to a subject. Forexample, biological activity of an antibody is retained if thebiological activity of the antibody in the pharmaceutical formulation iswithin about 30%, about 20%, or about 10% (within the errors of theassay) of the biological activity exhibited at the time thepharmaceutical formulation was prepared (e.g., as determined in anantigen binding assay).

Stability of a protein in an aqueous formulation may also be defined asthe percentage of monomer, aggregate, or fragment, or combinationsthereof, of the protein in the formulation. A protein “retains itsphysical stability” in a formulation if it shows substantially no signsof aggregation, precipitation and/or denaturation upon visualexamination of color and/or clarity, or as measured by UV lightscattering or by size exclusion chromatography. In one aspect of theinvention, a stable aqueous formulation is a formulation having lessthan about 10%, and preferably less than about 5% of the protein beingpresent as aggregate in the formulation.

Another characteristic of the aqueous formulation of the invention isthat, in some instances, diafiltering a protein using water results inan aqueous formulation having improved viscosity features in comparisonto the first protein solution (i.e., the viscosity of the diafilteredprotein solution is reduced in comparison to the first proteinsolution.) A person with skill in the art will recognize that multiplemethods for measuring viscosity can be used in the preparation offormulations in various embodiments of the invention. For example,kinematic viscosity data (cSt) may be generated using capillaries. Inother embodiments, dynamic viscosity data is stated, either alone orwith other viscosity data. The dynamic viscosity data may be generatedby multiplying the kinematic viscosity data by the density.

In one embodiment, the invention also provides a method for adjusting acertain characteristic, such as the osmolality and/or viscosity, asdesired in high protein concentration-water solutions, by addingnon-ionic excipients, such as mannitol, without changing other desiredfeatures, such as non-opalescence. As such, it is within the scope ofthe invention to include formulations which are water-based and havehigh concentrations of protein, where, either during or following thetransfer of the protein to water or during the course of thediafiltration, excipients are added which improve, for example, theosmolality or viscosity features of the formulation. Thus, it is alsowithin the scope of the invention that such non-ionic excipients couldbe added during the process of the transfer of the protein into thefinal low ionic formulation. Examples of non-ionizable excipients whichmay be added to the aqueous formulation of the invention for alteringdesired characteristics of the formulation include, but are not limitedto, mannitol, sorbitol, a non-ionic surfactant (e.g., polysorbate 20,polysorbate 40, polysorbate 60 or polysorbate 80), sucrose, trehalose,raffinose, and maltose.

The formulation herein may also contain more than one protein. Withrespect to pharmaceutical formulations, an additional, distinct proteinmay be added as necessary for the particular indication being treated,preferably those with complementary activities that do not adverselyaffect the other protein. For example, it may be desirable to providetwo or more antibodies which bind to TNF or IL-12 in a singleformulation. Furthermore, anti-TNF or anti-IL12 antibodies may becombined in the one formulation. Such proteins are suitably present incombination in amounts that are effective for the purpose intended.

Examples of proteins that may be included in the aqueous formulationinclude antibodies, or antigen-binding fragments thereof. Examples ofdifferent types of antibodies, or antigen-binding fragments thereof,that may be used in the invention include, but are not limited to, achimeric antibody, a human antibody, a humanized antibody, and a domainantibody (dAb). In one embodiment, the antibody used in the methods andcompositions of the invention is an anti-TNFα antibody, orantigen-binding portion thereof, or an anti-IL-12 antibody, or antigenbinding portion thereof. Additional examples of an antibody, orantigen-binding fragment thereof, that may be used in the inventionincludes, but is not limited to, 1D4.7 (anti-IL-12/anti-IL-23; AbbottLaboratories), 2.5(E)mg1 (anti-IL-18; Abbott Laboratories), 13C5.5(anti-Il-13; Abbott Laboratories), J695 (anti-IL-12; AbbottLaboratories), Afelimomab (Fab 2 anti-TNF; Abbott Laboratories), Humira(adalimumab (D2E7); Abbott Laboratories), Campath (Alemtuzumab),CEA-Scan Arcitumomab (fab fragment), Erbitux (Cetuximab), Herceptin(Trastuzumab), Myoscint (Imciromab Pentetate), ProstaScint (CapromabPendetide), Remicade (Infliximab), ReoPro (Abciximab), Rituxan(Rituximab), Simulect (Basiliximab), Synagis (Palivizumab), Verluma(Nofetumomab), Xolair (Omalizumab), Zenapax (Daclizumab), Zevalin(Ibritumomab Tiuxetan), Orthoclone OKT3 (Muromonab-CD3), Panorex(Edrecolomab), and Mylotarg (Gemtuzumab ozogamicin) golimumab(Centocor), Cimzia (Certolizumab pegol), Soliris (Eculizumab), CNTO 1275(ustekinumab), Vectibix (panitumumab), Bexxar (tositumomab and I¹³¹tositumomab) and Avastin (bevacizumab).

In one alternative, the protein is a therapeutic protein, including, butnot limited to, Pulmozyme (Dornase alfa), Regranex (Becaplermin),Activase (Alteplase), Aldurazyme (Laronidase), Amevive (Alefacept),Aranesp (Darbepoetin alfa), Becaplermin Concentrate, Betaseron(Interferon beta-1b), BOTOX (Botulinum Toxin Type A), Elitek(Rasburicase), Elspar (Asparaginase), Epogen (Epoetin alfa), Enbrel(Etanercept), Fabrazyme (Agalsidase beta), Infergen (Interferonalfacon-1), Intron A (Interferon alfa-2a), Kineret (Anakinra), MYOBLOC(Botulinum Toxin Type B), Neulasta (Pegfilgrastim), Neumega(Oprelvekin), Neupogen (Filgrastim), Ontak (Denileukin diftitox),PEGASYS (Peginterferon alfa-2a), Proleukin (Aldesleukin), Pulmozyme(Dornase alfa), Rebif (Interferon beta-1a), Regranex (Becaplermin),Retavase (Reteplase), Roferon-A (Interferon alfa-2), TNKase(Tenecteplase), and Xigris (Drotrecogin alfa), Arcalyst (Rilonacept),NPlate (Romiplostim), Mircera (methoxypolyethylene glycol-epoetin beta),Cinryze (C1 esterase inhibitor), Elaprase (idursulfase), Myozyme(alglucosidase alfa), Orencia (abatacept), Naglazyme (galsulfase),Kepivance (palifermin) and Actimmune (interferon gamma-1b).

Other examples of proteins which may be included in the methods andcompositions described herein, include mammalian proteins, includingrecombinant proteins thereof, such as, e.g., growth hormone, includinghuman growth hormone and bovine growth hormone; growth hormone releasingfactor; parathyroid hormone; thyroid stimulating hormone; lipoproteins;α-1-antitrypsin; insulin A-chain; insulin B-chain; proinsulin; folliclestimulating hormone; calcitonin; luteinizing hormone; glucagon; clottingfactors such as factor VIIIC, factor IX, tissue factor, and vonWillebrands factor; anti-clotting factors such as Protein C; atrialnatriuretic factor; lung surfactant; a plasminogen activator, such asurokinase or tissue-type plasminogen activator (t-PA); bombazine;thrombin; tumor necrosis factor-α and -β enkephalinase; RANTES(regulated on activation normally T-cell expressed and secreted); humanmacrophage inflammatory protein (MIP-1-α); serum albumin such as humanserum albumin; mullerian-inhibiting substance; relaxin A-chain; relaxinB-chain; prorelaxin; mouse gonadotropin-associated peptide; DNase;inhibin; activin; vascular endothelial growth factor (VEGF); receptorsfor hormones or growth factors; an integrin; protein A or D; rheumatoidfactors; a neurotrophic factor such as bone-derived neurotrophic factor(BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT4, NT-5, or NT-6), or anerve growth factor such as NGF-β; platelet-derived growth factor(PDGF); fibroblast growth factor such as aFGF and bFGF; epidermal growthfactor (EGF); transforming growth factor (TGF) such as TGFα and TGF-β,including TGF-β 1, TGF-β 2, TGF-β 3, TGF-β 4, or TGF-β 5; insulin-likegrowth factor-I and -II (IGF-I and IGF-II); des(1-3)-IGF-I (brainIGF-I); insulin-like growth factor binding proteins; CD proteins such asCD3, CD4, CD8, CD19 and CD20; erythropoietin (EPO); thrombopoietin(TPO); osteoinductive factors; immunotoxins; a bone morphogeneticprotein (BMP); an interferon such as interferon-α, -β, and -γ; colonystimulating factors (CSFs), e.g., M-CSF, GM-CSF, and G-CSF; interleukins(ILs), e.g., IL-1 to IL-10; superoxide dismutase; T-cell receptors;surface membrane proteins; decay accelerating factor (DAF); a viralantigen such as, for example, a portion of the AIDS envelope; transportproteins; homing receptors; addressins; regulatory proteins;immunoadhesins; antibodies; and biologically active fragments orvariants of any of the above-listed polypeptides.

IV. Uses of Invention

The formulations of the invention may be used both therapeutically,i.e., in vivo, or as reagents for in vitro or in situ purposes.

Therapeutic Uses

The methods of the invention may also be used to make a water-basedformulation having characteristics which are advantageous fortherapeutic use. The aqueous formulation may be used as a pharmaceuticalformulation to treat a disorder in a subject.

The formulation of the invention may be used to treat any disorder forwhich the therapeutic protein is appropriate for treating. A “disorder”is any condition that would benefit from treatment with the protein.This includes chronic and acute disorders or diseases including thosepathological conditions which predispose the mammal to the disorder inquestion. In the case of an anti-TNFα antibody, a therapeuticallyeffective amount of the antibody may be administered to treat anautoimmune disease, such as rheumatoid arthritis, an intestinaldisorder, such as Crohn's disease, a spondyloarthropathy, such asankylosing spondylitis, or a skin disorder, such as psoriasis. In thecase of an anti-IL-12 antibody, a therapeutically effective amount ofthe antibody may be administered to treat a neurological disorder, suchas multiple sclerosis, or a skin disorder, such as psoriasis. Otherexamples of disorders in which the formulation of the invention may beused to treat include cancer, including breast cancer, leukemia,lymphoma, and colon cancer.

The term “subject” is intended to include living organisms, e.g.,prokaryotes and eukaryotes. Examples of subjects include mammals, e.g.,humans, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits,rats, and transgenic non-human animals. In specific embodiments of theinvention, the subject is a human.

The term “treatment” refers to both therapeutic treatment andprophylactic or preventative measures. Those in need of treatmentinclude those already with the disorder, as well as those in which thedisorder is to be prevented.

The aqueous formulation may be administered to a mammal, including ahuman, in need of treatment in accordance with known methods ofadministration. Examples of methods of administration includeintravenous administration, such as a bolus or by continuous infusionover a period of time, intramuscular, intraperitoneal,intracerobrospinal, subcutaneous, intra-articular, intrasynovial,intrathecal, intradermal, transdermal, oral, topical, or inhalationadministration.

In one embodiment, the aqueous formulation is administered to the mammalby subcutaneous administration. For such purposes, the formulation maybe injected using a syringe, as well as other devices includinginjection devices (e.g., the Inject-ease and Genject devices); injectorpens (such as the GenPen); needleless devices (e.g., MediJector andBiojectorr 2000); and subcutaneous patch delivery systems. In oneembodiment, the device, e.g., a syringe, autoinjector pen, contains aneedle with a gauge ranging in size from 25 G or smaller in diameter. Inone embodiment, the needle gauge ranges in size from 25 G to 33 G(including ranges intermediate thereto, e.g., 25 sG, 26, 26 sG, 27 G, 28G, 29 G, 30 G, 31 G, 32 G, and 33 G). In a preferred embodiment, thesmallest needle diameter and appropriate length is chosen in accordancewith the viscosity characteristics of the formulation and the deviceused to deliver the formulation of the invention.

One advantage of the methods/compositions of the invention is that theyprovide large concentrations of a protein in a solution which may beideal for administering the protein to a subject using a needlelessdevice. Such a device allows for dispersion of the protein throughoutthe tissue of a subject without the need for an injection by a needle.Examples of needleless devices include, but are not limited to,Biojectorr 2000 (Bioject Medical Technologies), Cool. Click (BiojectMedical Technologies), Iject (Bioject Medical Technologies), Vitajet 3,(Bioject Medical Technologies), Mhi500 (The Medical House PLC), Injex 30(INJEX—Equidyne Systems), Injex 50 (INJEX—Equidyne Systems), Injex 100(INJEX-Equidyne Systems), Jet Syringe (INJEX—Equidyne Systems),Jetinjector (Becton-Dickinson), J-Tip (National Medical Devices, Inc.),Medi-Jector VISION (Antares Pharma), MED-JET (MIT Canada, Inc.),DermoJet (Akra Dermojet), Sonoprep (Sontra Medical Corp.), PenJet(PenJet Corp.), MicroPor (Altea Therapeutics), Zeneo (Crossject MedicalTechnology), Mini-Ject (Valeritas Inc.), ImplaJect (Caretek MedicalLTD), Intraject (Aradigm), and Serojet (Bioject Medical Technologies).

Also included in the invention are delivery devices that house theaqueous formulation. Examples of such devices include, but are notlimited to, a syringe, a pen (such as an autoinjector pen), an implant,an inhalation device, a needleless device, and a patch. An example of anautoinjection pen is described in U.S. application Ser. No. 11/824,516,filed Jun. 29, 2007.

The invention also includes methods of delivering the formulations ofthe invention by inhalation and inhalation devices containing saidformulation for such delivery. In one embodiment, the aqueousformulation is administered to a subject via inhalation using anebulizer or liquid inhaler. Generally, nebulizers use compressed air todeliver medicine as wet aerosol or mist for inhalation, and, therefore,require that the drug be soluble in water. Types of nebulizers includejet nebulizers (air-jet nebulizers and liquid-jet nebulizers) andultrasonic nebulizers.

Examples of nebulizers include Akita™ (Activaero GmbH) (seeUS2001037806, EP1258264). Akita™ is a table top nebulizer inhalationsystem (Wt: 7.5 kg, B×W×H: 260×170×270) based on Pari's LC Star thatprovides full control over patient's breathing pattern. The device candeliver as much as 500 mg drug in solution in less than 10 min with avery high delivery rates to the lung and the lung periphery. 65% of thenebulized particles are below 5 microns and the mass median aerodynamicdiameter (MMAD) is 3.8 microns at 1.8 bar. The minimum fill volume is 2mL and the maximum volume is 8 mL. The inspiratory flow (200 mL/sec) andnebulizer pressure (0.3-1.8 bar) are set by the smart card. The devicecan be individually adjusted for each patient on the basis of a lungfunction test.

Another example of a nebulizer which may be used with compositions ofthe invention includes the Aeroneb® Go/Pro/Lab nebulizers (AeroGen). TheAeroneb® nebulizer is based on OnQ™ technology, i.e., an electronicmicropump (⅜ inch in diameter and wafer-thin) comprised of a uniquedome-shaped aperture plate that contains over 1,000 precision-formedtapered holes, surrounded by a vibrational element. Aeroneb® Go is aportable unit for home use, whereas Aeroneb® Pro is a reusable andautoclavable device for use in hospital and ambulatory clinic, andAeroneb® Lab is a device for use in pre-clinical aerosol research andinhalation studies. The features of the systems include optimization andcustomization of aerosol droplet size; low-velocity aerosol deliverywith a precisely controlled droplet size, aiding targeted drug deliverywithin the respiratory system; flexibility of dosing; accommodation of acustom single dose ampoule containing a fixed volume of drug in solutionor suspension, or commercially available solutions for use in generalpurpose nebulizers; continuous, breath-activated or programmable; andadaptable to the needs of a broad range of patients, including childrenand the elderly; single or multi-patient use.

Aerocurrent™ (AerovertRx corp) may also be used with compositions of theinvention (see WO2006006963). This nebulizer is a portable, vibratingmesh nebulizer that features a disposable, pre-filled or user filleddrug cartridge.

Staccato™ (Alexza Pharma) may also be used with compositions of theinvention (see WO03095012). The key to Staccato™ technology isvaporization of a drug without thermal degradation, which is achieved byrapidly heating a thin film of the drug. In less than half a second, thedrug is heated to a temperature sufficient to convert the solid drugfilm into a vapor. The inhaler consists of three core components: aheating substrate, a thin film of drug coated on the substrate, and anairway through which the patient inhales. The inhaler is breath-actuatedwith maximum dose delivered to be 20-25 mg and MMAD in the 1-2 micronrange.

AERx® (Aradigm) may also be used with compositions of the invention (seeWO9848873, U.S. Pat. Nos. 5,469,750, 5,509,404, 5,522,385, 5,694,919,5,735,263, 5,855,564). AERx® is a hand held battery operated devicewhich utilizes a piston mechanism to expel formulation from the AERx®Strip. The device monitors patients inspiratory air flow and fires onlywhen optimal breathing pattern is achieved. The device can deliver about60% of the dose as emitted dose and 50-70% of the emitted dose into deeplung with <25% inter-subject variability.

Another example of a nebulizer device which may also be used withcompositions of the invention includes Respimat® (Boehringer). Respimat®is a multi-dose reservoir system that is primed by twisting the devicebase, which is compressed a spring and transfers a metered volume offormulation from the drug cartridge to the dosing chamber. When thedevice is actuated, the spring is released, which forces a micro-pistoninto the dosing chamber and pushes the solution through a uniblock; theuniblock consists of a filter structure with two fine outlet nozzlechannels. The MMAD generated by the Respimat® is 2 um, and the device issuitable for low dose drugs traditionally employed to treat respiratorydisorders.

Compositions of the invention may also be delivered using the CollegiumNebulizer™ (Collegium Pharma), which is a nebulizer system comprised ofdrug deposited on membrane. The dosage form is administered to a patientthrough oral or nasal inhalation using the Collegium Nebulizer afterreconstitution with a reconstituting solvent.

Another example of a nebulizer device which may also be used withcompositions of the invention includes the Inspiration® 626(Respironics). The 626 is a compressor based nebulizer for home care.The 626 delivers a particle size between 0.5 to 5 microns.

Nebulizers which can be used with compositions of the invention mayinclude Adaptive Aerosol Delivery® technology (Respironics), whichdelivers precise and reproducible inhaled drug doses to patientsregardless of the age, size or variability in breathing patterns of suchpatients. AAD® systems incorporate electronics and sensors within thehandpiece to monitor the patient's breathing pattern by detectingpressure changes during inspiration and expiration. The sensorsdetermine when to pulse the aerosol delivery of medication during thefirst part of inspiration. Throughout the treatment, the sensors monitorthe preceding three breaths and adapt to the patient's inspiratory andexpiratory pattern. Because AAD® systems only deliver medication whenthe patient is breathing through the mouthpiece, these devices allow thepatient to take breaks in therapy without medication waste. Examples ofAAD® system nebulizers include the HaloLite® AAD®, ProDose® AAD®, and1-Neb® AAD®.

The HaloLite® Adaptive Aerosol Delivery (AAD)® (Respironics) is apneumatic aerosolisation system powered by a portable compressor. TheAAD® technology monitors the patient's breathing pattern (typicallyevery ten milliseconds) and, depending upon the system being used,either releases pulses of aerosolized drug into specific parts of theinhalation, or calculates the dose drawn during inhalation from a“standing aerosol cloud” (see EP 0910421, incorporated by referenceherein).

The ProDos AAD® (Respironics) is a nebulizing system controlled by“ProDose Disc™” system. (Respironics). ProDos AAD® is a pneumaticaerosol system powered by a portable compressor, in which the dose to bedelivered is controlled by a microchip-containing disc inserted in thesystem that, among other things, instructs the system as to the dose todeliver. The ProDose Disc™ is a plastic disc containing a microchip,which is inserted into the ProDose AAD® System and instructs it as towhat dose to deliver, the number of doses, which may be deliveredtogether with various control data including drug batch code and expirydate (see EP1245244, incorporated by reference herein).

Promixin® can be delivered via Prodose AAD® for management ofPseudomonas aeruginosa lung infections, particularly in cystic fibrosis.Promixin® is supplied as a powder for nebulization that is reconstitutedprior to use.

The I-neb AAD® is a handheld AAD® system that delivers precise andreproducible drug doses into patients' breathing patterns without theneed for a separate compressor (“I-Neb”). The I-neb AAD® is aminiaturized AAD® inhaler based upon a combination of electronicmesh-based aerosolisation technology (Omron) and AAD® technology tocontrol dosing into patients' breathing patterns. The system isapproximately the size of a mobile telephone and weighs less than 8ounces. I-neb AAD® has been used for delivery of Ventavis® (iloprost)(CoTherix/Schering AG).

Another example of a nebulizer which may be used with compositions ofthe invention is Aria™ (Chrysalis). Aria is based on a capillary aerosolgeneration system. The aerosol is formed by pumping the drug formulationthrough a small, electrically heated capillary. Upon exiting thecapillary, the formulation rapidly cooled by ambient air to produce anaerosol with MMAD ranging from 0.5-2.0 um.

In addition the TouchSpray™ nebulizer (Odem) may be used to deliver acomposition of the invention. The TouchSpray™ nebulizer is a hand-helddevice which uses a perforate membrane, which vibrates at ultrasonicfrequencies, in contact with the reservoir fluid, to generate theaerosol cloud. The vibration action draws jets of fluid though the holesin the membrane, breaking the jets into drug cloud. The size of thedroplets is controlled by the shape/size of the holes as well as thesurface chemistry and composition of the drug solution. This device hasbeen reported to deliver 83% of the metered dose to the deep lung.Details of the TouchSpray™ nebulizer are described in U.S. Pat. No.6,659,364, incorporated by reference herein.

Additional nebulizers which may be used with compositions of theinvention include nebulizers which are portable units which maximizeaerosol output when the patient inhales and minimize aerosol output whenthe patient exhales using two one-way valves (see PARI nebulizers (PARIGmbH). Baffles allow particles of optimum size to leave the nebulizer.The result is a high percentage of particles in the respirable rangethat leads to improved drug delivery to the lungs. Such nebulizers maybe designed for specific patient populations, such a patients less thanthree years of age (PARI BABY™) and nebulizers for older patients (PARILC PLUS® and PARI LC STAR®).

An additional nebulizer which may be used with compositions of theinvention is the eFlow® nebulizer (PARI GmbH) which uses vibratingmembrane technology to aerosolize the drug solution, as well as thesuspensions or colloidal dispersions (TouchSpray™; ODEM (UnitedKingdom)). An e-Flow® nebulizer is capable of handling fluid volumesfrom 0.5 ml to 5 ml, and can produce aerosols with a very high densityof active drug, a precisely defined droplet size, and a high proportionof respirable droplets delivered in the shortest possible amount oftime. Drugs which have been delivered using the eFlow® nebulizer includeaztreonam and lidocaine. Additional details regarding the eFlow®nebulizer are described in U.S. Pat. No. 6,962,151, incorporated byreference herein.

Additional nebulizers which may be used with compositions of theinvention include a Microair® electronic nebulizer (Omron) and a Mystic™nebulizer (Ventaira). The Microair® nebulizer is extremely small anduses Vibrating Mesh Technology to efficiently deliver solutionmedications. The Microair device has 7 mL capacity and produces drugparticle MMAD size around 5 microns. For additional details regardingthe Microair® nebulizer see US patent publication no. 2004045547,incorporated by reference herein. The Mystic™ nebulizer uses strongelectric field to break liquid into a spray of nearly monodispersed,charged particles. The Mystic™ system includes a containment unit, adose metering system, aerosol generation nozzles, and voltage converterswhich together offer multi-dose or unit-dose delivery options. TheMystic™ device is breath activated, and has been used with Corus 1030™(lidocaine HCl), Resmycin® (doxorubicin hydrochloride), Acuair(fluticasone propionate), NCE with ViroPharm, and NCE with Pfizer.Additional details regarding the Mystic™ nebulizer may be found in U.S.Pat. No. 6,397,838, incorporated by reference herein.

Additional methods for pulmonary delivery of the formulation of theinvention are provided in U.S. application Ser. No. 12/217,972,incorporated by reference herein.

The appropriate dosage (“therapeutically effective amount”) of theprotein will depend, for example, on the condition to be treated, theseverity and course of the condition, whether the protein isadministered for preventive or therapeutic purposes, previous therapy,the patient's clinical history and response to the protein, the type ofprotein used, and the discretion of the attending physician. The proteinis suitably administered to the patient at one time or over a series oftreatments and may be administered to the patient at any time fromdiagnosis onwards. The protein may be administered as the sole treatmentor in conjunction with other drugs or therapies useful in treating thecondition in question.

The formulations of the invention overcome the common problem of proteinaggregation often associated with high concentrations of protein, and,therefore, provide a new means by which high levels of a therapeuticprotein may be administered to a patient. The high concentrationformulation of the invention provides an advantage in dosing where ahigher dose may be administered to a subject using a volume which isequal to or less than the formulation for standard treatment. Standardtreatment for a therapeutic protein is described on the label providedby the manufacturer of the protein. For example, in accordance with thelabel provided by the manufacturer, infliximab is administered for thetreatment of rheumatoid arthritis by reconstituting lyophilized proteinto a concentration of 10 mg/mL. The formulation of the invention maycomprise a high concentration of infliximab, where a high concentrationwould include a concentration higher than the standard 10 mg/mL. Inanother example, in accordance with the label provided by themanufacturer, Xolair (omalizumab) is administered for the treatment ofasthma by reconstituting lyophilized protein to a concentration of 125mg/mL. In this instance, the high concentration formulation of theinvention would include a concentration of the antibody omalizumab whichis greater than the standard 125 mg/mL.

Thus, in one embodiment, the formulation of the invention comprises ahigh concentration which is at least about 10%, at least about 20%, atleast about 30%, at least about 40%, at least about 50%, at least about60%, at least about 70%, at least about 80%, at least about 90%, atleast about 100%, at least about 110%, at least about 120%, at leastabout 130%, at least about 140%, at least about 150%, at least about175%, at least about 200%, at least about 225%, at least about 250%, atleast about 275%, at least about 300%, at least about 325%, at leastabout 350%, at least about 375%, at least about 400%, and so forth,greater than the concentration of a therapeutic protein in a known,standard formulation.

In another embodiment, the formulation of the invention comprises a highconcentration which is at least about 2 times greater than, at leastabout 3 times greater than, at least about 4 times greater than, atleast about 5 times greater than, at least about 6 times greater than,at least about 7 times greater than, at least about 8 times greaterthan, at least about 9 times greater than, at least about 10 timesgreater than and so forth, the concentration of a therapeutic protein ina known, standard formulation.

Characteristics of the aqueous formulation may be improved fortherapeutic use. For example, the viscosity of an antibody formulationmay be improved by subjecting an antibody protein solution todiafiltration using water without excipients as the diafiltrationmedium. As described above in Section II, excipients, such as thosewhich improve viscosity, may be added back to the aqueous formulationsuch that the final concentration of excipient is known and the specificcharacteristic of the formulation is improved for the specified use. Forexample, one of skill in the art will recognize that the desiredviscosity of a pharmaceutical formulation is dependent on the mode bywhich the formulation is being delivered, e.g., injected, inhaled,dermal absorption, and so forth. Often the desired viscosity balancesthe comfort of the subject in receiving the formulation and the dose ofthe protein in the formulation needed to have a therapeutic effect. Forexample, generally acceptable levels of viscosity for formulations beinginjected are viscosity levels of less than about 100 mPas,preferentially less than 75 mPas, even more preferentially less than 50mPas. As such, viscosity of the aqueous formulation may be acceptablefor therapeutic use, or may require addition of an excipient(s) toimprove the desired characteristic.

In one embodiment, the invention provides an aqueous formulationcomprising water and a human TNFα antibody, or antigen-binding portionthereof, wherein the formulation is excipient-free, wherein theformulation has viscosity which makes it advantageous for use as atherapeutic, e.g., low viscosity of less than 40 cP at 8° C., and lessthan 25 cP at 25° C. when the protein concentration is about 175 mg/mL.In one embodiment, the concentration of the antibody, or antigen-bindingportion thereof, in a formulation having improved viscosity is at leastabout 50 mg/mL. In one embodiment, the formulation of the invention hasa viscosity ranging between about 1 and about 2 mPas.

Non-Therapeutic Uses

The aqueous formulation of the invention may also be used fornon-therapeutic uses, i.e., in vitro purposes.

Aqueous formulations described herein may be used for diagnostic orexperimental methods in medicine and biotechnology, including, but notlimited to, use in genomics, proteomics, bioinformatics, cell culture,plant biology, and cell biology. For example, aqueous formulationsdescribed herein may be used to provide a protein needed as a molecularprobe in a labeling and detecting methods. An additional use for theformulations described herein is to provide supplements for cell culturereagents, including cell growth and protein production for manufacturingpurposes.

Aqueous formulations described herein could be used in protocols withreduced concern regarding how an excipient in the formulation may reactwith the experimental environment, e.g., interfere with another reagentbeing used in the protocol. In another example, aqueous formulationscontaining high concentrations of proteins may be used as a reagent forlaboratory use. Such highly concentrated forms of a protein would expandthe current limits of laboratory experiments.

Another alternative use for the formulation of the invention is toprovide additives to food products. Because the aqueous formulation ofthe invention consists essentially of water and protein, the formulationmay be used to deliver high concentrations of a desired protein, such asa nutritional supplement, to a food item. The aqueous formulation of theinvention provides a high concentration of the protein in water, withoutthe concern for excipients needed for stability/solubility which may notbe suitable for human consumption. For example, whey- and soy-derivedproteins are lending versatility to foods as these proteins have anability to mimic fat's mouthfeel and texture. As such, whey- andsoy-derived proteins may be added to foods to decrease the overall fatcontent, without sacrificing satisfaction. Thus, an aqueous formulationcomprising suitable amounts of whey- and soy-derived proteins may beformulated and used to supplement food products.

Articles of Manufacture

In another embodiment of the invention, an article of manufacture isprovided which contains the aqueous formulation of the present inventionand provides instructions for its use. The article of manufacturecomprises a container. Suitable containers include, for example,bottles, vials (e.g., dual chamber vials), syringes (such as dualchamber syringes), autoinjector pen containing a syringe, and testtubes. The container may be formed from a variety of materials such asglass, plastic or polycarbonate. The container holds the aqueousformulation and the label on, or associated with, the container mayindicate directions for use. For example, the label may indicate thatthe formulation is useful or intended for subcutaneous administration.The container holding the formulation may be a multi-use vial, whichallows for repeat administrations (e.g., from 2-6 administrations) ofthe aqueous formulation. The article of manufacture may further comprisea second container. The article of manufacture may further include othermaterials desirable from a commercial and user standpoint, includingother buffers, diluents, filters, needles, syringes, and package insertswith instructions for use.

The contents of all references, patents and published patentapplications cited throughout this application are incorporated hereinby reference

This invention is further illustrated by the following examples whichshould not be construed as limiting.

EXAMPLES

The following examples describe experiments relating to an aqueousformulation comprising water as the solution medium. It should be notedthat in some instances, decimal places are indicated using Europeandecimal notation. For example, in Table 31 the number “0,296” issynonymous with “0.296”.

Example 1: Diafiltration/Ultrafiltration with Adalimumab and J695

Materials and Methods

Adalimumab and J695 were diafiltered using pure water. After an at least5-fold volume exchange with pure water, the protein solutions wereultrafiltered to a final target concentration of at least 150 mg/mL.Osmolality, visual inspection and protein concentration measurements(OD280) were performed to monitor the status of the proteins duringDF/UF processing.

Size exclusion chromatography and ion exchange chromatography were usedto characterize protein stability in each final DF/UF product ascompared to the starting formulation, e.g., drug substance (DS) startingmaterial and protein standard. Drug substance or “DS” represents theactive pharmaceutical ingredient and generally refers to a therapeuticprotein in a common bulk solution.

-   -   Adalimumab Drug Substance, (Adalimumab extinction coefficient        280 nm: 1.39 mL/mg cm). Drug Substance did not contain        polysorbate 80. DS composition: 5.57 mM sodium phosphate        monobasic, 8.69 mM sodium phosphate dibasic, 106.69 mM sodium        chloride, 1.07 mM sodium citrate, 6.45 mM citric acid, 66.68 mM        mannitol.    -   Adalimumab solution used for dynamic light scattering (DLS)        measurements: Adalimumab solution that was diafiltered using        pure water as exchange medium was adjusted to 1 mg/mL        concentration by diluting the Adalimumab solution with Milli-Q        water and excipient stock solutions (excipients dissolved in        Milli-Q water), respectively.    -   J695 Drug Substance, (J695 extinction coefficient 280 nm: 1.42        mL/mg cm). DS composition: Histidine, Methionine, Mannitol, pH        5.8, and polysorbate 80.    -   Millipore Labscale™ Tangential Flow Filtration (TFF) system,        equipped with a 500 mL reservoir. The Labscale TFF system was        operated in discontinuous mode at ambient temperature according        to Millipore Operating Instructions. Stirrer speed was set to        approx. 1.5, and the pump speed was set to approximately 3. The        target inlet and outlet pressures were 15 mm psig approximately        50 mm psig, respectively.    -   Minimate™ Tangential Flow Filtration capsule, equipped with an        Omega™ PES membrane, 30 kDa cut-off. The capsule was rinsed for        30 min with 0.1 N NaOH and for another 30 min with Milli-Q        water.    -   780 pH meter, Metrohm, equipped with pH probe Pt1000, No.        6.0258.010, calibrated with buffer calibration solutions VWR, pH        4.00 buffer solution red, Cat. No. 34170-127, and pH 7.00 buffer        solution yellow, Cat. No. 34170-130.    -   Varian 50 Bio UV visible spectrophotometer, AI 9655, with a        fixed Cary 50 cell was used for protein concentration        measurements (280 nm wavelength). A 100 μL protein sample was        diluted with water (Milli-Q water for HPLC) to a final volume of        50.00 mL for protein concentration measurements of all J695        samples and the Adalimumab solution after DF/UF. Concentration        of all other Adalimumab samples was monitored by diluting 40 μL        sample solution with 1960 μL Milli-Q water. Disposable UV        cuvettes, 1.5 mL, semi-micro, Poly(methyl methacrylate) (PMMA),        were used for concentration measurements, Milli-Q water was used        as OD 280 blank.    -   Milli-Q water for HPLC grade was used as DF/UF medium.    -   A Malvern Zetasizer Nano ZS, Instrument No. AI 9494 was used for        DLS measurements.    -   Hellma precision cells, suprasil, Type No. 105.251-QS, light        path 3 mm, center 8.5, were used for DLS measurements (filled        with 75 μL sample, Malvern Mastersizer Nano ZS, Item No. AI        9494).    -   Knauer Osmometer Automatic, Instr. No. 83963, Berlin, Germany,        was used for osmolality measurement (calibrated with 400        mOsmol/kg NaCl calibration solution, Art. No. Y1241, Herbert        Knauer GmbH, Berlin, Germany).    -   250 mL Corning cell culture flasks, 75 cm², polystyrene,        sterile, Corning, N.Y., USA, were used for storage of the        protein solutions after the DF/UF operation.    -   Sodium chloride: J. T. Baker was used for preparing a 2 M NaCl        stock solution. The stock solution was used to prepare 1 mg/mL        Adalimumab solution in pure water with various concentrations of        NaCl (10, 20, 30, and 50 mM)    -   D-sorbitol, Sigma Chemical Co., St. Louis, Mo. 63178 was used        for preparing a 200 mg/mL sorbitol stock solution. The stock        solution was used to prepare 1 mg/mL Adalimumab solution in pure        water with various concentrations of sorbitol (10, 20, 30, and        40 mg/mL).        HPLC Methods    -   Adalimumab, SEC analysis: Sephadex 200 column (Pharmacia Cat.        No. 175175-01, S/N 0504057). Mobile phase 20 mM sodium        phosphate, 150 mM sodium chloride, pH 7.5, 0.5 mL/min flow rate,        ambient temperature, detection UV 214 nm and 280 nm. Each sample        was diluted to 1.0 mg/mL with Milli-Q water, sample injection        load 50 μg (duplicate injection).    -   Adalimumab, IEC analysis: Dionex, Propac WCX-10 column        (p/n 054993) along with a corresponding guard column (p/n        054994). Separation conditions: mobile phase A: 10 mM sodium        phosphate, pH 7.5; mobile phase B 10 mM Sodium phosphate, 500 mM        Sodium chloride, pH 5.5. 1.0 mL/min flow rate, ambient        temperature. Each sample was diluted to 1.0 mg/mL with Milli-Q        water, sample injection load 100 μg, duplicate injection.    -   J695, SEC analysis: Tosoh Bioscience G3000swxl, 7.8 mm×30 cm, 5        (Cat. No. 08541). Mobile phase 211 mM Na₂SO₄/92 mM Na₂HPO₄, pH        7.0. 0.3 mL/min flow rate, ambient temperature, detection UV 214        nm and 280 nm. Each sample was diluted to 2.5 mg/mL with Milli-Q        water, sample injection load 50 μg (duplicate injection).    -   J695, IEC analysis: Dionex, Propac WCX-10 column (p/n 054993)        along with a corresponding guard column (p/n 054994). Separation        conditions: mobile phase A: 10 mM Na₂HPO₄, pH 6.0; mobile phase        B 10 mM Na₂HPO₄, 500 mM NaCl, pH 6.0. 1.0 mL/min flow rate,        35° C. temperature. Each sample was diluted to 1.0 mg/mL with        Milli-Q water, sample injection load 100 μg. J695 Reference        standard 29001BF was run in triplicate as a comparison and was        diluted to 1 mg/ml in Milli-Q water based on the concentration        from the Certificate of Analysis.        Calculation of the Protein Concentration        Calculation Formula:

$E = {{{- l}{g\left( \frac{I}{I_{0}} \right)}} = {\left. {ɛ \cdot c \cdot d}\rightarrow c \right. = \frac{E}{ɛ \times d}}}$ɛ − absorption  coefficient c − concentrationd − length  of  cuvette  that  the  light  has  to  pass E − absorbanceI₀ − initial  light  intensityI − light  intensity  after  passing  through  sample$ɛ_{Adalimumab} = {1.39\frac{mL}{{mg} \times {cm}}}$$ɛ_{J\; 695} = {1.42\frac{mL}{{mg} \times {cm}}}$$ɛ_{HSA} = {1.042\frac{mL}{{mg} \times {cm}}}$1.1: DF/UF Processing of Adalimumab

DF/UF experiments are carried out following the standard operatingprocedures of the DF/UF equipment manufacturers. For example, theMillipore Labscale™ TFF system was equipped with a 500 mL reservoir andthe system operated in discontinuous mode at ambient temperature, inaccord with Millipore operating instructions. Stirrer speed was set toapproximately 1.5, and the pump speed was set to approximately 3. Thetarget inlet and outlet pressures were 15 mm psig and approximately 50mm psig, respectively, and the target pressures were monitored to ensurethat they were not exceeded.

A Minimate™ Tangential Flow Filtration capsule equipped with an Omega™PES membrane (Pall Corp., Port Washington, N.Y.), 30 kDa MWCO, was used.The capsule was rinsed for 30 min with 0.1 N NaOH and for another 30 minwith Milli-Q water before use.

Approximately 500 mL of Adalimumab solution were placed into the TFFreservoir and DF/UF processing was started in discontinuous mode. Table1 provides details on the In-Process-Control (IPC) data characterizingthe DF/UF process.

TABLE 1 Overview on Adalimumab DF/UF Processing Volume of Approx.Milli-Q volume Adalimumab Adalimumab water of Adalimumab concentrationconcentration Process added solution in of retentate Osmolality ofpermeate Step (mL) retentate (mL) (mg/mL) (mOsmol/kg) (mg/mL) 1 50054.66 305 — 2 400 68.33 297 3.15 3 300 — — — 4 250 550 43.73 169 1.39 5300 4.45 6 250 550 47.27 93 2.58 7 250 — — — 8 250 500 — — — 9 250 — — —10 250 500 — — — 11 250 — — — 12 250 500 52.24 9 1.24 13 300 90.27 7.5 —14 130 213.87 — 4.08 Fields filled with “—” indicate that no IPC sampleswere pulled at that step.

The DF/UF processing was stopped after an approximate 5-fold volumeexchange (1 volume exchange accounting for approx. 250 mL diafiltrationmedium). Assuming an ideal 100% excipient membrane permeability, thetheoretical final excipient concentration reached by the experimentparameters applied is C_(i)(250/500)⁵=0.03125*Ci, with Ci being theinitial concentration. The maximum excipient reduction was therefore96.875% (if constant volume diafiltration would have been used, thetheoretical excipient reduction with 5 diafiltration volumes would havebeen C_(i) e⁻⁵=0.00674, i.e. an approximate 99.3% maximum excipientreduction). Adalimumab solution was drained from the TFF system to a 250mL cell culture flask (low-volume rinse of the TFF system was performedusing WFI yielding a 175.05 mg/mL concentration; without the rinse, theretentate concentration was 213.87 mg/mL). Samples were pulled fordetermination of pH, osmolality and Adalimumab concentration.Additionally, samples were pulled for characterization by SEC and IEC.Characteristic parameters of the Adalimumab solution before and afterDF/UF processing, respectively, are listed in Table 2.

TABLE 2 Impact of DF/UF processing on Adalimumab solution solutionparameter before DF/UF solution after DF/UF pH 5.19 5.22 concentration(mg/mL) 54.66 175.05 osmolality (mOsmol/kg) 305 24 *SEC data (%aggregate, 0.26 00.50 monomer, 99.74 99.50 fragment) 0.00 0.00 *IEC data(acidic regions, lys 13.89 14.07 0, 62.05 61.97 lys 1, 19.14 18.51 lys2, %) 4.83 4.73 *samples were subjected to one freeze/thaw step (−80°C./25° C.) before analysis via SEC and IEC

In the course of DF/UF processing, Adalimumab concentration exceeded 210mg/mL. Throughout the experiment, the protein solution remained clear,and no solution haziness or protein precipitation, which would haveindicated Adalimumab solubility limitations, was observed. Compared tothe original Adalimumab DS solution (˜55 mg/mL), Adalimumab solutiondiafiltered by using pure water as DF/UF exchange medium revealed loweropalescence, despite a more than 3-fold increase in proteinconcentration (˜175 mg/mL).

1.2: Adalimumab Characterization Via Chromatography

FIG. 1 shows a SEC chromatogram of an Adalimumab reference standard(Adalimumab standard (bottom line)) compared to the Adalimumab drugstandard solution before (middle line) and after (top line) the DF/UFprocessing procedure. Note that all samples were frozen at −80° C. priorto analysis.

Table 3 also contains the IEC chromatogram data (note all samples werefrozen at −80° C. prior to analysis).

TABLE 3 IEC Data of Various Adalimumab Samples % Acidic % Acidic SampleName Region 1 Region 2 % 0 Lys % 1 Lys % 2 Lys Reference standard 2.6911.66 60.77 19.42 5.40 Adalimumab DS 2.51 11.38 62.05 19.14 4.83Adalimumab, after 2.26 11.81 61.97 18.51 4.73 DF/UF1.3: Impact of Excipients on Adalimumab Hydrodynamic Diameter (D_(h))

It was previously determined that the hydrodynamic diameter of J695, asdetermined by dynamic light scattering (DLS) measurements, was notablydecreased when formulating J695 into pure water. J695 in WFI had a D_(h)of ˜3 nm, far below the values that are expected for immunoglobulins.Upon addition of low amounts of ionizable NaCl, the Dh values increasedto ˜10 nm (independent of the NaCl concentration). Addition ofnon-ionizable mannitol increased J695 solution osmolality, but had noeffect on J695 Dh.

In order to assess the impact of excipients on the hydrodynamic diameterof Adalimumab that had been processed according to the above DF/UFprocedure, the Adalimumab solution from the DF/UF experiment was used toformulate Adalimumab solutions in pure water, but with varying levels ofNaCl (0-50 mM) and sorbitol (0-40 mg/mL), respectively. The impact ofsorbitol (a non-ionizable excipient) and NaCl (ionizable excipient)concentrations on Dh of Adalimumab monomer is displayed in FIG. 2.

The hydrodynamic diameter of Adalimumab monomer in pure water was 2.65nm. The Adalimumab Dh response to salt and non-ionic excipients wasidentical to the J695 response seen previously. Adalimumab Dh wasvirtually not impacted by the presence of sorbitol. Low concentrationsof NaCl induced the monomer hydrodynamic diameter to increase toexpected levels of ˜11 nm. These findings demonstrate that proteinhydrodynamic diameters as measured by dynamic light scattering arecrucially impacted by the presence of ionizable excipients. Absence ofionizable excipients also is linked to solution viscosities.

These findings have implications for high-concentrated proteinsolutions: the lower the hydrodynamic diameter, the lower the spatialvolume proteins occupy. In high-concentration scenarios, the viscositiesof protein solutions that are prepared by using water as DF/UF exchangemedium will be substantially lower than the viscosities of traditionalprotein formulations containing considerable amounts of ionizable bufferexcipients. The Adalimumab data confirmed this, as viscosities of 200mg/mL Adalimumab solutions in water for injection were found to be wellbelow 50 mPas, independent of pH (e.g. pH 4, pH 5, and pH 6). More dataon the effect of pH on D_(h) can be found in Example 17 below.

Overall, these findings are useful in high-concentration proteinformulation activities, where viscosity related manufacturing anddosing/delivery problems are well known. Furthermore, these findingsshow that the osmolality values of final Drug Product can be adjustedwith non-ionizable excipients such as sugars or sugar alcohols asdesired without inducing an increase in protein Dh and solutionviscosity, respectively.

1.4: DF/UF Processing of J695 (Anti-IL12 Antibody)

Approximately 200 mL of J695 solution were adjusted to pH 4.4 with 1 Mphosphoric acid and filled into the TFF reservoir (pH adjustment wasmade to ensure a positive zetapotential of J695 monomers and thus avoida potential impact of uncharged protein monomer on data). Then, 300 mLof Milli-Q water were added to the TFF reservoir, and DF/UF processingwas started in discontinuous mode. 250 mL reservoir volume, 250 mL ofMilli-Q water were added, and DF/UF processing was started again. TheDF/UF processing was stopped after a total of 5 volume exchange stepswere performed (1 volume exchange accounting for approx. 250 mL).

Assuming an ideal 100% excipient membrane permeability, the theoreticalfinal excipient concentration reached by the experiment parametersapplied is Ci(250/500)5=0.03125*Ci, with Ci being the initialconcentration. The maximum excipient reduction is therefore 96.875% (ifconstant volume diafiltration would have been used, the theoreticalexcipient reduction with 5 diafiltration volumes would have been Cie-5=0.00674, i.e. an approx. 99.3% maximum excipient reduction). J695solution was drained from the TFF system to a 250 mL cell culture flask(no rinse of the TFF system was performed). Samples were pulled fordetermination of pH, osmolality and J695 concentration. Additionally,samples were pulled for characterization by SEC and IEC. Characteristicparameters of the J695 solution before and after DF/UF processing,respectively, are listed in Table 4.

TABLE 4 Impact of DF/UF Processing on J695 Solution parameter solutionbefore DF/UF solution after DF/UF pH 4.40 4.70 concentration (mg/mL)122.9 192.8 osmolality (mOsmol/kg) 265 40 SEC data (% aggregate, 0.410.69 monomer, 98.42 98.11 fragment) 1.18 1.21 IEC data 92.00 92.11 (sumof isoforms, 5.17 5.30 acidic species, 2.83 2.59 basic species, %)

As with Adalimumab, the DF/UF experiments on J695 substantiate theprincipal possibility of processing and formulating J695 by using purewater as exchange medium in DF/UF operations. Both SEC and IEC datasuggest no substantial impact on J695 stability while DF/UF processingfor an overall period of 1.5 days (process interruption overnight) atambient temperature using Milli-Q water as diafiltration medium.Throughout the experiment, the protein solution remained clear,indicating no potential J695 solubility limitations.

1.5: J695 Characterization

Table 5 describes the percentages for aggregate, monomer and fragmentcontent for the three solutions as determined by SEC chromatogram.

TABLE 5 Data from SEC chromatogram % Aggregate % Monomer % FragmentSample Name content content content Reference standard 0.45 98.00 1.56J695 before DF/UF 0.41 98.42 1.18 J695 after 0.69 98.11 1.21 DF/UF

FIG. 3 shows the IEC profile of J695 reference standard (bottom graph)and J695 DS, pH adjusted to pH 4.4 (top graph).

Only a small increase in aggregate content was observed in the J695samples after DF/UF processing.

FIG. 4 shows the IEC profile of J695 after DF/UF with Milli-Q water, pH4.7 (top graph), and J695 DS before DF/UF, pH adjusted to pH 4.4 (bottomcurve).

As depicted in FIG. 4, the DF/UF step had no notable impact on J695stability when monitored by IEC. The differences between the two J695reference standards (refer to FIG. 3) can be attributed to differencesin the manufacturing processes between the 3000 L and 6000 L DScampaigns. Table 6 highlights more details on IEC data.

TABLE 6 IEC Data of Various J695 Samples Sample Name 0 glu (1) otherisoforms acidic basic Reference standard 43.57 50.06 4.47 1.90 J69535.74 56.26 5.17 2.83 J695 after DF/UF 36.59 55.52 5.30 2.591.6: Conclusion

The above example provides a diafiltration/ultrafiltration (DF/UF)experiment where water (Milli-Q water for HPLC) was used asdiafiltration medium for monoclonal antibodies Adalimumab and J695.

Adalimumab was subjected to DF/UF processing by using pure water asDF/UF exchange medium and was formulated at pH 5.2 at highconcentrations (>200 mg/mL) without inducing solution haziness, severeopalescence or turbidity formation. Upon one subsequent freeze/thawstep, SEC and IEC data suggested no notable difference betweenAdalimumab solution formulated in water via DF/UF processing and theoriginal Adalimumab DS.

J695 also was also subjected to DF/UF processing by using pure water asDF/UF exchange medium and was formulated at pH 4.7 without impactingJ695 stability (visual inspection, SEC, IEC).

When formulated using such a DF/UF processing, the hydrodynamic diameter(Dh) of Adalimumab monomer was approx. 2.65 nm. The presence ofnon-ionic excipients such as sorbitol in concentrations up to 40 mg/mLwas shown to have no impact on Dh data, whereas ionic excipients such asNaCl already in low concentrations were demonstrated to induce theAdalimumab monomer Dh to increase to approx. 11 nm (such Dh data arecommonly monitored for IgG). Similar findings were made earlier forJ695.

In conclusion, processing and formulating proteins using pure water asDF/UF exchange medium is feasible. Assuming an ideal 100% excipientmembrane permeability, the theoretical final excipient concentrationreached by the constant volume diafiltration with 5 diafiltrationvolumes would be Ci e-5=0.00674, i.e. an approx. 99.3% maximum excipientreduction. Using 6 diafiltration volume exchanges, an theoretical 99.98%maximum excipient reduction would result.

Examples 2 to 5 describe experimental execution with respect to threedifferent proteins which were concentrated into an aqueous formulation,and Examples 6 to 11 describe analysis of each of the aqueousformulations.

Materials and Methods for Examples 2-11

-   -   Adalimumab protein solution (10 mg/mL) in water for injection,        Protein Drug Substance (DS) Material (49.68 mg/mL), DS contains        Tween 80, Adalimumab, Adalimumab Drug Product (DP) (40 mg,        solution for injection, filtered solution from commercial        production). Protein absorption coefficient 280 nm: 1.39.    -   J695 protein solution (10 mg/mL) in water for injection, Protein        Drug Substance (DS) (54 mg/mL), DS contains Tween 80. Absorption        coefficient 280 nm: 1.42    -   HSA protein solution (10 mg/mL) in water for injection, DP        without Tween 80, Grifols Human Serum Albumin Grifols®, 20%, 50        mL. Absorption coefficient 280 nm: 1.042    -   6 R vial and 10R vial    -   vial preparation: the vials were washed and autoclaved    -   stoppers, 19 mm, West, 4110/40/grey    -   sample repositories (e.g., Eppendorf sample repository,        Safe-Lock or simple snap-fit, 1-2 mL)    -   single-use syringes, sterile, 20 mL; NormJect, 10 mL    -   single use filter units (filter Millex®-GV, Syringe Driven        Filter Unit, PVDF 0.22 μm; Millex®-GP, Syringe Driven Filter        Unit, PES 0.22 μm, Sterivex®0.22 μm Filter Unit)    -   Vivaspin concentrators (cut off 10 kDa, PES; cut off 3 kDa, PES)    -   Pipettes (e.g., Eppendorf, max.: 1000 μL)    -   Water for injection    -   Centrifuge (Eppendorf) and Centrifuge No. 1        (temperature-controlled)    -   Diafiltration equipment: Millipore Labscale™ TFF System,        Millipore diafiltration membrane: Adalimumab: Polyethersulfone;        J695: Polyethersulfone; HSA: regenerated cellulose    -   pH probe (Metrohm pH-Meter, protein-suitable probe, biotrode 46)    -   Laminar-Air-Flow bench, Steag LF-Werkbank, Mp.-No. 12.5    -   NaCl; mannitol    -   Gemini 150 Peltier Plate Rheometer, Malvern    -   Rheometer MCR 301 [temperature-controlled P-PTD 200 (plate with        Peltier tempering)] and cone/plate measurement system CP50/0.5°        Ti as well as CP50/1° (stainless steel); Anton Paar    -   Capillary viscometer, Schott, capillaries: type 537 20, type 537        10, type 537 13    -   1 M hydrochloric acid (J. T. Baker)        Analytics    -   UV/VIS spectrophotometry (OD 280 nm); Photon Correlation        Spectroscopy (PCS): for approximately 10 mg/mL and approximately        20 mg/mL: 1.1 mPas, 3 runs, 30 s, one measurement, 25° C., from        approximately 30 mg/mL and above: 1.9 mPas, 30 s, 3 runs, one        measurement, 25° C.    -   Size Exclusion Chromatography (SEC) and Ion Exchange        Chromatography (IEC), as described below.    -   viscosity measurement: different viscometers with individual and        different set-ups were used        Calculation of the Protein Concentration        Calculation Formula:

$E = {{{- l}{g\left( \frac{I}{I_{0}} \right)}} = {\left. {ɛ \cdot c \cdot d}\rightarrow c \right. = \frac{E}{ɛ \times d}}}$ɛ − absorption  coefficient c − concentrationd − length  of  cuvette  that  the  light  has  to  pass E − absorbanceI₀ − initial  light  intensityI − light  intensity  after  passing  through  sample$ɛ_{Adalimumab} = {{1.39\frac{mL}{{mg} \times {cm}}ɛ_{J\; 695}} = {{1.42\frac{mL}{{mg} \times {cm}}ɛ_{HSA}} = {1.042\frac{mL}{{mg} \times {cm}}}}}$Viscosity Data and Calculation for AdalimumabAdalimumab commercial formulation (approximately 194 mg/mL) density:

${\rho = 1},{05475\frac{g}{{cm}^{3}}}$Adalimumab commercial formulation (approximately 194 mg/mL) kinematicviscosity:

K − constant  of  the  capillaryt − the  time  the  solution  needs  for  passing  the  capillary  [s]v − kinematic  viscosity${v = {{K \times t} = 0}},{03159\frac{{mm}^{2}}{s^{2}} \times 279},{{36\mspace{14mu} s} = 8},{825\frac{{mm}^{2}}{s}}$Adalimumab commercial formulation (approximately 194 mg/mL) dynamicviscosity:

η − dynamic  viscosity p − density${\eta = {{v \times \rho} = 8}},{825\frac{{mm}^{2}}{s} \times 1},{{05475\frac{g}{{cm}^{3}}} = \underset{\_}{9,{308\mspace{14mu}{mPas}}}}$Viscosity Data and Calculation for Human Serum AlbuminHSA commercial formulation (approximately 200 mg/mL) density:

${\rho = 1},{05833\frac{g}{{cm}^{3}}}$HSA  commerical  formulation(app.  200  mg/mL)  kinematic  viscosity:K − constant  of  the  capillaryt − the  time  the  solution  needs  for  passing  the  capillary  [s]v − kinematic  viscosity${v = {{K \times t} = 0}},{01024\frac{{mm}^{2}}{s^{2}} \times 337},{{69\mspace{20mu} s} = \underset{\_}{3,{46\frac{{mm}^{2}}{s}}}}$HSA commercial formulation (approximately 200 mg/mL) dynamic viscosity:

η − dynamic  viscosity p − density${\eta = {{v \times \rho} = 3}},{46\frac{{mm}^{2}}{\text{s}} \times 1},{{05833\frac{g}{{cm}^{3}}} = \underset{\_}{3,{662\mspace{14mu}{mPas}}}}$HSA  in  WFI  (approximately  180  mg/mL)  density:${\rho = 1},{07905\frac{g}{{cm}^{3}}}$HSA in WFI (approximately 180 mg/mL) kinematic viscosity:

K − constant  of  the  capillaryt − the  time  the  solution  needs  for  passing  the  capillary  [s]v − kinematic  viscosity${v = {{K \times t} = 0}},{09573\frac{{mm}^{2}}{s^{2}} \times 185},{{3\mspace{14mu} s} = \underset{\_}{17,{72\frac{{mm}^{2}}{s}}}}$HSA in WFI (approximately 180 mg/mL) dynamic viscosity:

η − dynamic  viscosity p − density${\eta = {{v \times \rho} = {17}}},{72\frac{{mm}^{2}}{s} \times 1},{{07905\frac{g}{{cm}^{3}}} = \underset{\_}{19,{121\mspace{14mu}{mPas}}}}$General Experimental Execution for Arriving at High ConcentrationFormulation

Generally, the process of the invention for arriving at a highconcentration, salt-free, protein formulation includes diafiltration ofthe initial drug substance material, followed by a procedure to increasethe concentration of the drug substance in the solution. This may bedone in separate procedures or may be done in separate or coincidingsteps within the same procedure.

Diafiltration

A sufficient amount of Drug Substance (DS) material (depending onprotein concentration of DS) was subjected to diafiltration. Prior todiafiltration, the DS material was diluted with water for injection ˜10mg/ml. Note that in total approximately 540 mL of a 10 mg/mL solutionwas needed for the experiment.

Water for injection was used as diafiltration medium. The number ofdiafiltration steps performed was 5 to 7 (one diafiltration step equalsone total volume exchange). In-Process-Control (IPC) samples were pulledprior to diafiltration and after diafiltration step (200 μL forosmolality, 120 μL for SEC).

Diafiltration with TFF equipment is performed by applying the followingparameters:

stirrer: position 2

pump: position 2

pressure up-stream/inlet: max 20-30 psi

pressure down-stream/outlet: max 10 psi

(Parameters used in this experiment were derived from manufacturer'srecommendations. One with skill in the art would be able to alter theparameters of equipment operation to accommodate a particular protein orvariances in equipment, formulation, viscosity, and other variables.)

After diafiltration, protein concentration was assessed by means ofOD280. If protein concentration was >10 mg/mL, the protein concentrationwas adjusted to 10 mg/mL by appropriately diluting the solution withwater for injection.

Concentration

20 mL of diafiltrated protein solution (e.g., Adalimumab, J695, HSA)were put into a Vivaspin 20 Concentrator. The concentrator was closedand put into the centrifuge. The protein solution was centrifuged atmaximum speed (5000 rpm).

Sample Pulls

Samples of the concentrated solutions were pulled at: 10 mg/mL and thenevery 10 mg/mL (20, 30, 40 mg/ml etc.) or until the protein aggregatesvisibly, and samples were analyzed as follows:

-   -   The protein solution was homogenized in the Vivaspin        concentrator and filled in an adequate vial.    -   Optical appearance was inspected directly in the vial.    -   300 μL was used for UV spectroscopy, 160 μL for PCS, 120 μL for        SEC and 300 μL for IEX.    -   the samples for SEC and IEX were stored at −80° C.        Dynamic Light Scattering (DLS) Protocol

Dynamic light scattering was performed using the Zetasizer Nano ZS(Malvern Instruments, Southborough, Mass.) equipped with Hellmaprecision cells (Plainview, N.Y.), suprasil quartz, type 105.251-QS,light path 3 mm, center Z8.5 mm, with at least 60 μL sample fill, usingprotein samples as is and placed directly in measurement cell. Prior tomeasurement, the cell window was checked to verify that the solution wasfree of air bubbles or particles/dust/other contaminants that may impactDLS measurement. Measurements were taken under standard operatingprocedures (“general purpose” mode, 25° C., refractive index set to1.45, measurement mode set to “manual”, 1 run per measurement, eachcomprising 3 measurements of 30 s each, type of measurement set to“size”). Dispersion Technology Software, version 4.10b1, MalvernInstruments, was used to analyze data. About 70 μL of a sample solutionwere filled in precision cell for analysis of hydrodynamic diameters(Dh). Default sample viscosity was set 1.1 mPas for low concentratedprotein solutions (e.g. <5 mg/mL). Underlying measurement principlesconcluded that minimal differences between real viscosity values of thesample solution to be measured and the use of default viscosities doesnot impact DLS data readout substantially. This was verified byperforming DLS measurements of low protein concentration solutions (<5mg/mL) where solution viscosities were determined and taken into accountin subsequent DLS measurements. For all samples with higher proteinconcentration, viscosities were determined and taken into account duringDLS measurements.

Example 2: Formulation Comprising High TNFα Antibody Concentration

2.1: Diafiltration

Prior to diafiltration, Adalimumab (49.68 mg/mL) was diluted with waterfor injection to a concentration of approximately 15 mg/mL. Therefore140.8 mL Adalimumab solution (49.68 mg/mL) were filled in a 500 mLvolumetric flask. The flask was filled up to the calibration mark withwater for injection. The volumetric flask was closed and gently shakenfor homogenization of the solution. The TFF labscale system was flushedwith water. Then the membrane (PES) was adapted and was also flushedwith 1 L distilled water. Next, the TFF labscale system and the membranewere flushed with approximately 300 mL of water for injection. Thediluted Adalimumab solution was then filled in the reservoir of the TFF.A sample for an osmolality measurement (300 μL), UV spectrophotometry(500 μL) and a sample for SEC analysis (120 μL) were pulled. The systemwas closed and diafiltration was started. The DF/UF(diafiltration/ultrafiltration) was finished after 5 volume exchangesand after an osmolality value of 3 mosmol/kg was reached. The pH-valueof the Adalimumab solution after diafiltration was pH 5.25.

Diafiltration with TFF equipment was performed by applying the followingparameters:

stirrer: position 2

pump: position 2

pressure up-stream/inlet: max 20-30 psi

pressure down-stream/outlet: max 10 psi

After diafiltration, protein concentration was assessed by means ofOD280. The concentration was determined to be 13.29 mg/mL.

The Adalimumab solution was sterile filtered.

The TFF and the membrane were flushed with approximately 1 L distilledwater and then with 500 mL 0.1 M NaOH. The membrane was stored in 0.1 MNaOH, the TFF was again flushed with approximately 500 mL distilledwater.

2.2: Protein Concentration

Prior to concentrating the antibody, the protein concentration was againassessed by means of OD280. Adalimumab concentration was determined tobe 13.3 mg/mL. The Adalimumab solution was then diluted to 10 mg/mL.375.94 mL of Adalimumab solution (13.3 mg/mL) was filled in a 500 mLvolumetric flask and the flask was filled up to the calibration markwith water for injection (WFI). 75.19 mL of Adalimumab solution (13.3mg/mL) was also filled in a 100 mL volumetric flask, and filled up tothe calibration mark with pure water, i.e., water for injection (WFI).Both flasks were gently shaken for homogenization. The solutions fromboth flasks were placed in a 1 L PETG bottle. The bottle was gentlyshaken for homogenization.

Four Vivaspin 20 concentrators (10 kDa) were used. In three Vivaspins,20 mL of Adalimumab solution (10 mg/mL) were filled (in each). In thefourth Vivaspin device, water was filled as counterbalance weight whilecentrifuging. The concentrators were closed and put into the centrifuge.The Adalimumab solution was centrifuged applying 4500×g centrifugationforce (in a swing out rotor).

2.3: Sample Pull

Samples of the concentrated Adalimumab solution were pulled when theyreached a concentration of 10 mg/mL and at each subsequent 10 mg/mLconcentration increment increase (at 20 mg/mL, 30 mg/mL, 40 mg/mL etc.until approximately 200 mg/mL). At 40 minute intervals, theconcentrators were taken out of the centrifuge, the solution washomogenized, and the solution and the centrifuge adapters were cooledfor approximately 10 min on ice. After each 10 mg/mL concentratingincrement, the solutions in the concentrators were homogenized, theoptical appearance was checked and samples were pulled for analysis viaUV (300 μL), PCS (160 μL), SEC (120 μL) and IEC (300 μL). After samplepulls, the concentrators were filled up to approximately 20 mL withAdalimumab solution (10 mg/mL).

Visual Inspection and PCS analysis of protein precipitation were used todetermine the solubility limit of Adalimumab protein (i.e. isoforms) inthe solution.

At a concentration of approximately 80 mg/mL it became obvious that theAdalimumab solution was not opalescent anymore, opalescence being aknown characteristic of Adalimumab solutions having a high amount offragment. Therefore, it was suspected that fragmentation might haveoccurred during experiment execution. For further analysis, a sample ofAdalimumab solution (approximately 80 mg/mL) was analyzed by SEC. Theremainder of the solution in each Vivaspin, as well as the rest ofAdalimumab solution (10 mg/mL), was removed to 50 mL falcon tubes andstored at −80° C. The SEC analysis showed a purity of 99.6% monomer.

The solution was thawed in water bath at 25° C. and sterile filtered.Afterwards the solutions from 3 falcon tubes were place into eachVivaspin. The concentrators were filled to approximately 20 mL and theconcentration was continued. The experiment was finished as aconcentration of approximately 200 mg/mL was reached.

All SEC and IEC samples were stored at −80° C. until further analysis.UV and PCS were measured directly after sample pull. The rest of theconcentrated Adalimumab solution was placed in Eppendorf repositoriesand stored at −80° C.

Table 7 shown below describes calculation of volumes of protein solutionto be refilled into the concentrators while concentrating Adalimumabsolution. The scheme was calculated before experiment execution todefine at which volume samples have to be pulled. The duration ofcentrifugation is shown in Table 8.

TABLE 7 Centrifugation Scheme volume concentration 10 mg/ml step volume(ml) (mg/ml) protein solution 0 20 10 conc. 1 10 20 sampling 2 9 20filling 3 20 14.5 11 conc. 4 9.66 30 sampling 5 8.66 30 filling 6 2018.66 11.34 conc. 7 9.33 40 sampling 8 8.33 40 filling 9 20 22.49 11.67conc. 10 8.99 50 sampling 11 7.99 50 filling 12 20 25.98 12.01 conc. 138.66 60 sampling 14 7.66 60 filling 15 20 29.15 12.34 conc. 16 8.32 70sampling 17 7.32 70 filling 18 20 31.96 12.68 conc. 19 7.99 80 sampling20 6.66 80 filling 21 20 34.46 13.01 conc. 22 7.65 90 sampling 23 6.6590 filling 24 20 36.6 13.35 conc. 25 7.32 100 sampling 26 6.32 100filling 27 20 38.44 13.68 conc. 28 6.98 110 sampling 29 5.98 110 filling30 20 39.9 14.02 conc. 31 6.65 120 sampling 32 5.65 120 filling 33 2041.07 14.35 conc. 34 6.31 130 sampling 35 5.31 130 filling 36 20 41.8614.69 conc. 37 5.98 140 sampling 38 4.98 140 conc. 39 4.64 150 154.14174.14

TABLE 8 Centrifugation times required for concentrating the Adalimumabsolution concentration [from -> to] [mg/ml] time [min] 10 to 20 15 20 to30 20 30 to 40 27 40 to 50 30 50 to 60 40 60 to 70 50 70 to 80 60 80 to90 67  90 to 100 80 100 to 110 100 110 to 200 206Results from the concentration of Adalimumab are also shown below inTable 12.2.4: Viscosity Measurement

Adalimumab solutions comprising either 50 mg/mL in WFI or 200 mg/mL inWFI were measured for viscosity. 50 mg/mL and 200 mg/mL in WFI weremeasured using a Gemini 150 Peltier Plate Rheometer, Malvern, and the200 mg/mL in WFI solution was also measured via rheometer MCR 301[temperature-controlled P-PTD 200 (plate with Peltier tempering)] andcone/plate measurement system CP50/1 (stainless steel); Anton Paar).

Adalimumab solutions (200 mg/mL) in repository tubes were thawed andhomogenized in a 6R vial. 1 mL Adalimumab (200 mg/mL) was diluted with 3mL WFI to obtain the diluted solution (for a 50 mg/ml Adalimumabsolution).

For the rheometer Gemini 150 approximately 2 mL were needed and for theMCR 301 less than 1 mL was needed for measurement.

Adalimumab (approximately 194 mg/mL) in the commercial formulation wasobtained by using Vivaspin tubes. The tubes were filled with Adalimumabsolution in commercial buffer and centrifugation was applied until a 194mg/mL concentration was reached. Viscosity was measured with thecapillary viscometer Schott.

2.5: Summary

In sum, Adalimumab was concentrated from 50 mg/mL to approximately 194mg/mL in Vivaspin 20 tubes in four different tubes. At the beginning, 20mL of Adalimumab solution (50 mg/mL) were in every tube (four tubes). Atthe end of the concentration, 5 mL of Adalimumab solution (approximately194 mg/mL) were in every tube. The concentration step was performed at5000 rpm (approximately 4500 g). After every hour, the oblong beakersand the protein solution in the Vivaspin tubes were cooled in crushedice for approximately 10 to 15 min. The density was measured withdensity measurement device DMA 4500, Anton Paar. Further analysis of thehigh Adalimumab concentration formulation is provided in Examples 5 to11.

Example 3: Formulation Comprising High Concentration 11-12 Antibody

3.1: Diafiltration

Prior to diafiltration, IL-12 antibody J695 (54 mg/mL) was diluted withwater for injection to a concentration of approximately 15 mg/mL. Thiswas done by placing 150 mL J695 solution (54 mg/mL) in a 500 mLvolumetric flask and filling the flask to the calibration mark withwater for injection. The volumetric flask was closed and gently shakenfor homogenization of the solution. The TFF labscale system was flushedwith water. Then the polyethersulfone membrane (PES) was adapted and wasalso flushed with 1 L of distilled water. Afterwards the TFF labscalesystem and the membrane were flushed with approximately 300 mL of waterfor injection. Next, the diluted J695 solution was placed in thereservoir of the TFF. A sample for osmolality measurement (300 UVspectrophotometry (500 μL) and a sample for SEC analysis (120 μL) werepulled. The system was closed and diafiltration was started. After 200mL of processing the DF volume, the diafiltration was stopped andanother sample for UV measurement was pulled. The DF/UF was stoppedafter 1800 mL diafiltration volume (approximately factor 3.5 volumeexchange), reaching an osmolality value of 4 mosmol/kg. The pH-value ofthe J695 solution after diafiltration was pH 6.48.

Diafiltration with TFF equipment was performed by applying the followingparameters:

stirrer: position 2

pump: position 2

pressure up-stream/inlet: max 20-30 psi

pressure down-stream/outlet: max 10 psi

After diafiltration, the protein concentration was assessed by means ofOD280. The concentration was determined to be 16.63 mg/mL.

The J695 solution was sterile filtered.

The TFF instrument and the membrane were flushed with approximately 1 Lof distilled water and then with 500 mL 0.1 M NaOH. The membrane wasstored in 0.1 M NaOH and the TFF was again flushed with approximately500 mL of distilled water.

3.2: Concentrating

Prior to concentrating, the J695 solution was diluted to 10 mg/mL: 316mL of J695 solution (16.63 mg/mL) was placed in a 500 mL volumetricflask and the flask was filled to the calibration mark with water forinjection (WFI). Additionally, 64 mL of J695 solution (16.63 mL) wasplaced in a 100 mL volumetric flask and filled to the calibration markwith WFI. Both flasks were gently shaken for homogenization. Thesolutions from both flasks were placed in a 1 L PETG bottle. The bottlewas gently shaken for homogenization.

Four Vivaspin 20 concentrators (10 kDa cut-off) were used. 20 mL of J695solution (10 mg/mL) were place in each of three Vivaspins. In the fourthVivaspin concentrator device, water was filled as counterbalance weightwhile centrifuging. The concentrators were closed and put into thecentrifuge. The J695 solution was centrifuged applying 4500×gcentrifugation force (in a swing out rotor).

3.3: Sample Pull

Samples of the concentrated J695 solution were pulled when they reacheda concentration of 10 mg/mL and at each subsequent 10 mg/mLconcentration increment increase (at 20 mg/mL, 30 mg/mL, 40 mg/mL etc.until 200 mg/mL). After every 40 minutes, the concentrators were takenout of the centrifuge, the solution was homogenized, and the solutionand the centrifuge adapters were cooled for approximately 10 min on ice.After every 10 mg/mL concentration increase, the solutions in theconcentrators were homogenized, the optical appearance was checked andsamples were pulled for UV (300 PCS (160 μL), SEC (120 μL) and IECanalysis (300 μL). After sample pulls, the concentrators were filled upto approximately 20 mL with J695 solution (10 mg/mL).

Visual Inspection and PCS analysis were used to determine the solubility(i.e., to check for potential precipitation) and stability of J695.

At the conclusion of the experiment, a concentration of approximately200 mg/mL was reached.

All SEC and IEC samples were stored at −80° C. for further analysis (seebelow). UV spectrophotometry and PCS measurements were taken directlyafter each sample pull. The rest of the concentrated J695 solution wasplaced in Eppendorf repositories and stored at −80° C.

Details regarding the centrifugation scheme are provided above in Table7. The duration of the J695 centrifugation are provided in Table 9.

TABLE 9 Centrifugation Times used to Concentrate the J695 Solutionconcentration [from -> to] [mg/mL] time [min] 10 to 20 13 20 to 30 22 30to 40 27 40 to 50 38 50 to 60 45 60 to 70 80 70 to 80 90 80 to 90 105 90 to 100 165 100 to 200 2703.4: Impact of Excipients on the Hydrodynamic Diameter of J695

In this experiment the impact of sodium chloride and mannitol,separately, on the hydrodynamic diameter of J695 was analyzed. For thispurpose, stock solutions of sodium chloride (12 mg/mL) and of mannitol(120 mg/mL) were prepared. 1.2 g NaCl was weighed in a beaker, which wasfilled with approximately 70 mL of WFI, and 12.002 g of Mannitol wasweighed in a beaker which was filled with approximately 70 mL of WFI.The two solutions were stirred for homogenization. Each solution wasplaced in a volumetric flask, which was filled to the calibration markwith WFI. The flasks were gently shaken for homogenization.

Approximately 8 mL of J695 solution (approximately 200 mg/mL) was thawedat 37° C. The solution was filled in a 10R vial and homogenized. Seven2R vials were filled with 500 μL J695 solution (approximately 200 mg/mL)each. The filling scheme is described in Table 10 below.

TABLE 10 Filling Scheme for Preparation of J695 Solutions ContainingDifferent Concentrations of NaCl or Mannitol vol. NaCl vol. mannitolvolume stock stock ABT-874 solution solution vol. concentration (200 (12mg/ml) (12 mg/mL) WFI excipient excipient mg/ml( [μL] [μL] [μL] [μL] — —500 — — 500 NaCl  2 mg/mL 500 167 — 333 NaCl  4 mg/mL 500 333 — 167 NaCl 6 mg/mL 500 500 — — mannitol 20 mg/mL 500 — 167 333 mannitol 40 mg/mL500 — 333 167 mannitol 60 mg/mL 500 — 500 —

The 2R vials were gently homogenized via shaking. Thereafter, PCS andosmolality measurements were taken of the different J695 solutions (100mg/mL).

To prepare samples for PCS analysis, the cuvettes were first flushedwith 50 μL of the sample. Then measurements were taken using 100 μL ofthe sample.

Further analysis of the high J695 concentration formulation is providedin Examples 5 to 11.

Example 4: High Concentration Human Serum Albumin (HSA) Formulation

4.1: Diafiltration

Prior to diafiltration, HSA solution (200 mg/mL, commercial formulation)was diluted with water for injection to a concentration of 15.29 mg/mL.To achieve this, 38 mL HSA (200 mg/mL) were filled in a 500 mLvolumetric flask. The flask was filled to the calibration mark withwater for injection. The volumetric flask was closed and gently shakenfor homogenization of the solution. The TFF labscale system was flushedwith water. Then the membrane (regenerated cellulose) was adapted andwas also flushed with 1 L of distilled water. Afterwards the TFFlabscale system and the membrane were flushed with approximately 300 mLwater for injection. Next, the diluted HSA solution was filled in thereservoir of the TFF. Samples for osmolality measurement (300 μL), UVspectrophotometry (500 μL) and a sample for SEC analysis (120 μL) werepulled. The system was closed and diafiltration was started. Afterdiafiltration of approximately 300 mL of volume, a UV measurement of thepermeate was taken. The permeate revealed a concentration of 2.74 mg/mL,indicating that protein was passing through the membrane. Thediafiltration was stopped after approximately 500 mL of DF, and anothersample for UV measurement was pulled (HSA concentration 11.03 mg/mL).The DF/UF was finished after 950 mL of diafiltration volume(approximately 2 volume exchanges) and after reaching an osmolalityvalue of 4 mosmol/kg. The pH-value of the HSA solution afterdiafiltration was pH 7.13.

UV spectrophotometric measurement of the permeate was performed threetimes (n=3).

Diafiltration with TFF equipment was performed by applying the followingparameters:

stirrer: position 2

pump: position 2

pressure up-stream/inlet: max 20-30 psi

pressure down-stream/outlet: max 10 psi

After diafiltration, protein concentration was assessed by means ofOD280. The concentration was determined 9.41 mg/mL.

The HSA solution was sterile filtered. The TFF and the membrane wereflushed with approximately 1 L of distilled water. Afterwards anintegrity test was done (see Operating Instructions Labscale™ TFFSystem, page 5-3 to 5-5, 1997). The volume flow was 1.2 mL/min, thus,the integrity test was passed (acceptable maximal limit 3 mL/min). Themembrane was once more flushed with 500 mL of distilled water and thenwith 500 mL of 0.05 M NaOH. The membrane was stored in 0.05 M NaOH, theTFF was again flushed with approximately 500 mL of distilled water.

4.2: Concentration Process

Prior to concentrating the HSA protein solution, the concentration wasassessed by means of OD280 and was determined to be 9.52 mg/mL. FourVivaspin 20 concentrators (10 kDa) were used. 20 mL of HSA solution(9.52 mg/mL) were placed in each of 3 Vivaspin concentrators. In thefourth Vivaspin, water was filled as counterweight balance whilecentrifuging. The concentrators were closed and put into the centrifuge.The HSA solution was centrifuged applying 4500×g centrifugation force(in a swing out rotor).

4.3: Sample Pull

Samples of the concentrated HSA solution were pulled when theconcentration reached 10 mg/mL and subsequently after every 10 mg/mLconcentration increment increase (at 20 mg/mL, 30 mg/mL, 40 mg/mL etc.until approximately 180 mg/mL). Every 40 minutes the concentrators weretaken out of the centrifuge, the solution was homogenized, and thesolution and the centrifuge adapters were cooled for approximately 10min on ice. After every 10 mg/mL concentration increment increase, thesolutions in the concentrators were homogenized, the optical appearancewas checked and samples were pulled for analysis via UV (300 μL), PCS(160 μL), SEC (120 μL) and IEC (300 μL). After the sample pull, HSAsolution (9.52 mg/mL) was added to the concentrators, up toapproximately 20 mL.

When the projected concentration for the HSA solution in theconcentrator reached approximately 20 mg/mL, permeate was measured viaOD280, revealing a concentration of 0.5964 mg/mL. The concentration ofthe HSA solution was only 15.99 mg/mL, which was less than expected. Asample of concentrated HSA in WFI (10 mg/mL) was analyzed via SEC toscrutinize for potential fragmentation. The HSA solution (15.99 mg/mL)in the Vivaspins was placed in falcon tubes and stored at −80° C. Theremainder of the original HSA solution (9.52 mg/mL) used to fill theconcentrators was also stored at −80° C.

SEC analysis was performed to determine whether the HSA proteinunderwent degradation, producing small fragments that could pass throughthe membrane. The SEC analysis, however, revealed a monomer amount of92.45% for 10 mg/mL HSA in WFI with virtually no fragments.

The solutions that were stored at −80° C. were thawed at 25° C. andsterile filtered. The solutions in the falcon tubes were transferred inone Vivaspin 20 concentrator each (3 kDa cut-off). The Vivaspins werefilled up with HSA solution (9.52 mg/mL) and centrifuged (refer to 3.2concentration process described above).

Visual Inspection and PCS analysis were used to determine the solubilitylimit of HSA.

At the completion of the experiment, a concentration of approximately180 mg/mL HSA was reached.

All SEC and IEC samples were stored at −80° C. until further analysis.UV and PCS measurements were performed directly after sample pull. Therest of the concentrated HSA solution was placed in Eppendorfrepositories and stored at −80° C.

An overview of the centrifugation scheme is described above in Table 7.The duration of the centrifugation used to concentrate HSA is describedin Table 11.

TABLE 11 Centrifugation Times Necessary to Concentrate HSA Solutionconcentration [from ->to] [mg/mL] time [min] 10 to 20 9 20 to 30 30 30to 40 40 40 to 50 50 50 to 60 80 60 to 70 90 70 to 80 110 80 to 90 130 90 to 100 170 100 to 180 3604.4: Impact of the pH Value on the Hydrodynamic Diameter of HSA

The following part of the experiment was performed to evaluate apotential impact of pH on the hydrodynamic diameter of HSA when theprotein is dissolved in WFI. Four 6R vials were filled with 5.09 mL HSAsolution (9.83 mg/mL), and pH values from 3 to 6 were set up with 1 MHCl (actual pH: 3.04, 3.99, 5.05, 6.01). These solutions were eachtransferred to a separate 10 mL volumetric flask. The flasks were thenfilled to the calibration mark and gently shaken for homogenization.

The HCl solutions were placed in 10R vials and analyzed via PCS. Thesolutions were sterile filtered and measured again via PCS. Also, 5.09mL HSA solution (9.83 mg/mL) were transferred in a 10 mL volumetricflask and this was filled with WFI to the calibration mark. The flaskwas gently shaken for homogenization and then the solution was sterilefiltered and measured via PCS.

Sample Preparation for PCS Measurement:

The cuvettes were flushed with 50 μL of sample. Measurement wasperformed with 100 μL of sample volume.

4.5: Viscosity Measurement

For HSA in commercial formulation (200 mg/mL) and for HSA in WFI(approximately 180 mg/mL) the viscosity was measured with a capillaryviscometer (Schott, MP.-No. 33.2).

A 15 mL aliquot was pulled from a 50 mL bottle of commercial formulationHSA. HSA in WFI was thawed at approximately 20° C. and approximately 9mL were aliquotted in a Falcon tube. The density was measured withdensity measurement device DMA 4500, Anton Paar.

Further analysis of the high HSA concentration formulation is providedin Examples 5 to 11.

Example 5: Analysis of High Protein Formulations—Optical Appearance

In contrast to Adalimumab in the commercial formulation, Adalimumab inWFI did not reveal opalescence. J695 also did not reveal any opalescencephenomena when dissolved in WFI. Despite the fact that the proteinconcentration of Adalimumab was 80 mg/mL and 200 mg/mL in WFI, there wasvirtually no opalescence observed. In contrast, the commercialformulation comprising 50 mg/mL Adalimumab revealed notable opalescencein the commercial formulation. Thus, the use of pure water, i.e., WFI,as a dissolution medium had a positive effect on protein solutionopalescence.

It was a surprising observation that (in addition to being soluble atall at such a high protein concentration) Adalimumab in WFI appeared tohave a low viscosity, even at higher concentrations such as 200 mg/mL.

Depending on the concentration, the optical characteristics/color of HSAsolutions changed from clear and slightly yellow (10 mg/mL in WFI) toclear and yellow (approximately 180 mg/mL in WFI).

During the concentration process, no precipitation was observed for theAdalimumab solution and HSA solution. Precipitation would have been anindication for solubility limitations. The solutions stayed clear untilthe experiment was finalized.

It is to be highlighted that the experiments were not finished becausepotential solubility limits were approached and precipitation occurred,but were finished because the solution volumes remaining in theconcentrators were not sufficient to proceed with concentrating (i.e.lack of material). It appears very likely that the solubility limits ofAdalimumab, J695, and HSA are well beyond 220 mg/mL.

In the J695 solution a crystal like precipitate was observed when thehigh-concentrated solution was stored over night at 2-8° C. in theconcentrators (approximately 120 mg/mL). The crystal like precipitateredissolved after approximately 3-5 min when the solution was stored atambient temperature. Thus the environment created by dissolving J695 athigh concentration in pure water provides conditions where proteincrystallization might be performed by mere temperature cycling (e.g.,from ambient temperature to 2-8° C.).

Example 6: Analysis of High Protein Formulations—Protein Concentration

The calculation of the protein concentrations is provided above in theMaterials and Methods section.

An overview of the concentration of Adalimumab, J695, and HSA into purewater, high protein formulation is provided below in Tables 12-14:

TABLE 12 Concentrations of Adalimumab as Assessed Via OD280 during theConcentration Process aver- absorb- age dilu- concen- sample name ancevalue tion tration Adalimumab in WFI 10 mg/ml 0.680 0.650 20 9.35 0.6950.575 Adalimumab in WFI 20 mg/ml 1 1.064 0.813 40 23.40 0.688 0.687Adalimumab in WFI 20 mg/ml 2 0.781 0.788 40 22.68 0.793 0.791 Adalimumabin WFI 20 mg/ml 3 0.870 0.824 40 23.71 0.883 0.719 Adalimumab in WFI 30mg/ml 1 0.817 0.807 60 34.84 0.812 0.793 Adalimumab in WFI 30 mg/ml 20.839 0.827 60 35.69 0.813 0.829 Adalimumab in WFI 30 mg/ml 3 0.7700.744 60 32.10 0.729 0.732 Adalimumab in WFI 40 mg/ml 1 0.494 0.491 10035.35 0.493 0.488 Adalimumab in WFI 40 mg/ml 2 0.499 0.501 100 36.060.516 0.489 Adalimumab in WFI 40 mg/ml 3 0.495 0.512 100 36.81 0.5230.517 Adalimumab in WFI 50 mg/ml 1 0.574 0.585 100 42.11 0.579 0.603Adalimumab in WFI 50 mg/ml 2 0.671 0.634 100 45.63 0.630 0.601Adalimumab in WFI 50 mg/ml 3 0.579 0.574 100 41.27 0.574 0.568Adalimumab in WFI 60 mg/ml 1 0.838 0.837 100 60.21 0.833 0.840Adalimumab in WFI 60 mg/ml 2 0.793 0.777 100 55.89 0.767 0.770Adalimumab in WFI 60 mg/ml 3 0.802 0.780 100 56.10 0.759 0.779Adalimumab in WFI 70 mg/ml 1 0.911 0.878 100 63.15 0.866 0.857Adalimumab in WFI 70 mg/ml 2 1.012 0.996 100 71.68 0.976 1.001Adalimumab in WFI 70 mg/ml 3 0.879 0.871 100 62.66 0.874 0.861Adalimumab in WFI 80 mg/ml 1 0.512 0.510 200 73.45 0.489 0.531Adalimumab in WFI 80 mg/ml 2 0.542 0.526 200 75.64 0.519 0.517Adalimumab in WFI 80 mg/ml 3 0.551 0.531 200 76.42 0.511 0.531Adalimumab in WFI 90 mg/ml 1 0.550 0.547 200 78.64 0.550 0.539Adalimumab in WFI 90 mg/ml 2 0.549 0.548 200 78.80 0.551 0.543Adalimumab in WFI 90 mg/ml 3 0.532 0.534 200 76.81 0.533 0.537Adalimumab in WFI 100 mg/ml 1 0.640 0.628 200 90.36 0.621 0.623Adalimumab in WFI 100 mg/ml 2 0.748 0.747 200 107.41 0.735 0.757Adalimumab in WFI 100 mg/ml 3 0.625 0.621 200 89.39 0.616 0.623Adalimumab in WFI 110 mg/ml 1 0.674 0.669 200 96.19 0.671 0.660Adalimumab in WFI 110 mg/ml 2 0.693 0.668 200 96.05 0.690 0.620Adalimumab in WFI 110 mg/ml 3 0.604 0.640 200 92.05 0.664 0.652Adalimumab in WFI 200 mg/ml 1 0.863 0.698 400 201.00 0.612 0.621Adalimumab in WFI 200 mg/ml 2 1.055 0.791 400 227.53 0.658 0.659Adalimumab in WFI 200 mg/ml 3 0.732 0.665 400 191.44 0.648 0.615

TABLE 13 Concentrations of J695 as Assessed Via OD280 during theConcentration Process average concen- sample name absorbance valuedilution tration ABT-874 in WFI 10 mg/ml 0.715 0.703 20 9.90 0.708 0.705ABT-874 in WFI 20 mg/ml 1 0.686 — 40 19.31 ABT-874 in WFI 20 mg/ml 20.684 — 40 19.26 ABT-874 in WFI 20 mg/ml 3 0.685 — 40 19.29 ABT-874 inWFI 30 mg/ml 1 0.700 — 60 29.59 ABT-874 in WFI 30 mg/ml 2 0.703 — 6029.70 ABT-874 in WFI 30 mg/ml 3 0.684 — 60 28.91 ABT-874 in WFI 40 mg/ml1 0.539 — 100 37.97 ABT-874 in WFI 40 mg/ml 2 0.540 — 100 38.02 ABT-874in WFI 40 mg/ml 3 0.520 — 100 36.65 ABT-874 in WFI 50 mg/ml 1 0.698 —100 49.15 ABT-874 in WFI 50 mg/ml 2 0.653 — 100 45.95 ABT-874 in WFI 50mg/ml 3 0.623 — 100 43.87 ABT-874 in WFI 60 mg/ml 1 0.834 — 100 58.75ABT-874 in WFI 60 mg/ml 2 0.781 — 100 55.02 ABT-874 in WFI 60 mg/ml 30.778 — 100 54.76 ABT-874 in WFI70 mg/ml 1 1.103 — 100 77.69 ABT-874 inWFI 70 mg/ml 2 1.102 — 100 77.62 ABT-874 in WFI 70 mg/ml 3 1.110 — 10078.13 ABT-874 in WFI 80 mg/ml 1 0.671 — 200 94.45 ABT-874 in WFI 80mg/ml 2 0.746 — 200 105.06 ABT-874 in WFI 80 mg/ml 3 0.664 — 200 93.45ABT-874 in WFI 90 mg/ml 1 0.826 — 200 116.37 ABT-874 in WFI 90 mg/ml 20.809 — 200 113.92 ABT-874 in WFI 90 mg/ml 3 0.804 — 200 113.27 ABT-874in WFI 100 mg/ml 1 0.861 — 200 121.21 ABT-874 in WFI 100 mg/ml 2 0.993 —200 139.80 ABT-874 in WFI 100 mg/ml 3 0.985 — 200 138.73 ABT-874 in WFI200 mg/ml 1 0.681 0.805 400 226.67 0.864 0.869 ABT-874 in WFI 200 mg/ml2 0.690 0.767 400 216.10 0.828 0.784 ABT-874 in WFI 200 mg/ml 3 0.7080.745 400 209.83 0.789 0.738Tables 14a and 14b: Concentrations of HSA as Assessed Via OD280 duringthe Concentration Process

TABLE 14a sample name absorbance dilution concentration HSA in WFI 10mq/ml 0.515  20 9.88 HSA in WFI 30 mg/ml 1 0.398  60 22.94 HSA in WFI 30mq/ml 2 0.395  60 22.73 HSA in WFI 30 mg/ml 3 0.400  60 23.00 HSA in WFI40 mg/ml 1 0.383 100 36.78 HSA in WFI 40 mg/ml 2 0.389 100 37.33 HSA inWFI 40 mg/ml 3 0.368 100 35.29 HSA in WFI 50 mq/ml 1 0.479 100 45.97 HSAin WFI 50 mg/ml 2 0.496 100 47.61 HSA in WFI 50 mq/ml 3 0.465 100 44.61HSA in WFI 60 mg/ml 1 0.609 100 58.47 HSA in WFI 60 mg/ml 2 0.653 10062.69 HSA in WFI 60 mg/ml 3 0.568 100 54.52 HSA in WFI 70 mg/ml 1 0.645100 61.89 HSA in WFI 70 mg/ml 2 0.623 100 59.76 HSA in WFI 70 mg/ml 30.618 100 59.28 HSA in WFI 80 mq/ml 1 0.393 200 75.37 HSA in WFI 80mg/ml 2 0.436 200 83.69 HSA in WFI 80 mg/ml 3 0.363 200 69.67 HSA in WFI90 mg/ml 1 0.484 200 92.90 HSA in WFI 90 mg/ml 2 0.439 200 84.22 HSA inWFI 90 mq/ml 3 0.419 200 80.50 HSA in WFI 100 mg/mL 1 0.604 200 115.93HSA in WFI 100 mg/mL 2 0.573 200 110.00 HSA in WFI 100 mg/mL 3 0.585 200112.30

TABLE 14b average sample name absorbance value dilution concentrationHSA in WFI 180 mg/ml 1 0.946 0.952 200 182.79 0.950 0.961 HSA in WFI 180mg/ml 2 0.994 0.929 200 178.24 0.906 0.886 HSA in WFI 180 mg/ml 3 0.8430.896 200 172.05 0.963 0.884

All three proteins evaluated remained soluble in the concentrationranges evaluated (i.e. >200 mg/mL for Adalimumab and J695, >175 mg/mLfor HSA). No indications of insolubility, e.g., the clouding phenomenaor precipitation occurring in the solution, were observed. ForAdalimumab, the results indicate that, over the concentration rangeevaluated, all Adalimumab isoforms (i.e. lysine variants) remainedsoluble, as no precipitation occurred at all. This observation is alsoconsistent with ion exchange chromatography data described in Example11, which describes that the sum of lysine variants stayed virtuallyconsistent regardless of Adalimumab concentration.

Example 7: Analysis of High Protein Formulations—Viscosity

7.1: Adalimumab Viscosity

The viscosity of Adalimumab (approximately 50 mg/mL) in water forinjection was determined to be around 1.5-2 mPas. For Adalimumab(approximately 200 mg/mL) in WFI, two values were determined. The onevalue determined with cone/plate rheometer from Malvern (Gemini 150) wasapproximately 6-6.5 mPas, while the other value (measured withcone/plate rheometer from Anton Paar, MCR 301) was approximately 12mPas.

Adalimumab commercial formulation (approximately 194 mg/mL) viscosity:

K—constant of the capillary

t—the time the solution needs for passing the capillary [s]

ν—kinematic viscosity

η—dynamic viscosity

ρ—density

time [s] K [mm2/s2] v [mm2/s] ρ [g/cm3] η [mPas] 279.36 0.03159 8.8251.05475 9.31

The viscosity of Adalimumab in WFI (approximately 200 mg/mL) wasdetermined to be approximately 12 mPas with the viscosimeter from AntonPaar and approximately 6 mPas determined with the viscometer fromMalvern. In contrast, the viscosity of Adalimumab in the commercialformulation (approximately 194 mg/mL) is higher, at 9.308 mPas (measuredwith the capillary viscometer from Schott).

7.2: Human Serum Albumin Viscosity

HSA commercial formulation (approximately 200 mg/mL) viscosity:

K—constant of the capillary

t—the time the solution needs for running through the capillary [s]

ν—kinematic viscosity

η—dynamic viscosity

ρ—density

time [s] K [mm2/s2] v [mm2/s] ρ [g/cm3] η [mPas] 337.69 0.01024 3.461.05475 3.66HSA in WFI (approximately 180 mg/mL) viscosity:K—constant of the capillaryt—the time the solution needs for running through the capillary [s]ν—kinematic viscosityη—dynamic viscosityρ—density

time [s] K [mm2/s2] v [mm2/s] ρ [g/cm3] η [mPas] 185.3 0.09573 17.721.07905 19.12

The viscosity of HSA in WFI (approximately 180 mg/mL) was determined tobe approximately 19.121 mPas. The viscosity of HSA in the commercialformulation (approximately 194 mg/mL) was determined to be 9.308 mPas(measured with the capillary viscometer from Schott).

7.3: Analysis of the Viscosities of Adalimumab and HSA

The dynamic viscosity of Adalimumab 50 mg/mL in WFI was lower than theviscosity of Adalimumab 200 mg/mL in WFI and in commercial buffer,respectively. For HSA the dynamic viscosity for a concentration of 180mg/mL in WFI was about six-fold higher than for a concentration of 200mg/mL in commercial buffer. Thus, it seems that the intensity ofviscosity change (i.e. increase and decrease, respectively) due toeffects conveyed by pure water as dissolution medium may depend on theindividual protein.

Example 8: Analysis of the Hydrodynamic Diameters of High ProteinFormulations—Photon Correlation Spectroscopy (PCS)

The following example provides an analysis of the hydrodynamic diameter(Dh) (the z-average of the mean hydrodynamic molecule diameter) ofvarious proteins in aqueous formulations obtained using the DF/UFmethods of the invention.

8.1: Adalimumab Hydrodynamic Diameter

As shown in FIGS. 5 and 6, a trend can be observed where thehydrodynamic diameter (D_(h)) increases with increasing Adalimumabconcentration. FIG. 5 shows the correlation between hydrodynamicdiameter (z-average) and the concentration of Adalimumab in WFI. FIG. 6shows the correlation between hydrodynamic diameter (peak monomer) andthe concentration of Adalimumab in WFI.

The difference between the D_(h) determined from the 23.27 mg/mL samplecompared to the 34.20 mg/mL sample exists because of assumptions made inthe Standard Operating Procedure (SOP) for hydrodynamic diametermeasurement. For Adalimumab samples having ≤23.27 mg/mL concentration,PCS measurements were performed with a SOP that assumes a 1.1 mPas valuefor the viscosity of the samples. For Adalimumab samples having ≥34.20mPas, a SOP assuming a 1.9 mPas sample viscosity was used. It is knownthat PCS data are strongly influenced by the given viscosity of thesample solution, as PCS data is based on random Brownian motion of thesample specimen, which is impacted by sample viscosity. Thus, theincrease in the hydrodynamic diameter with increasing proteinconcentration can be explained, as increasing protein concentrationraises the viscosity of the solution (higher viscosity leads to lowerBrownian motion and higher calculated D_(h) data). The protein moleculesexperience a lower random Brownian motion and thus, for a givenviscosity, the hydrodynamic diameters of the sample specimen arecalculated higher. Overall, the z-average based D_(h) values and theD_(h) values of the monomer match well. Additionally, no increase inD_(h) indicative for protein insolubility is observed with increasingconcentration (i.e. high molecular weight aggregates and precipitate (ifpresent) would induce a substantial increase in D_(h)).

8.2: J695 Hydrodynamic Diameter

FIGS. 7 and 8 show that the hydrodynamic diameter of J695 was relativelyindependent from the protein concentration until a 114.52 mg/mLconcentration was reached. Increasing the J695 concentration from 114.52mg/mL to 133.25 mg/mL induced a rapid increase in D_(h). Thehydrodynamic diameter at the 217.53 mg/mL concentration was higher thanat 114.52 mg/mL. This finding was not surprising as both proteinsolutions were measured using the same SOP (assuming same viscosity of1.9 mPas), when in reality the viscosity increases as the proteinconcentration increases. The strong increase from 114.52 mg/mL to 133.25mg/mL thus can be explained as an artifact.

8.3: Human Serum Albumin Hydrodynamic Diameter

The hydrodynamic diameter of HSA in WFI was found to decrease asconcentrations rose from 9.88 mg/mL to 112.74 mg/mL. From 112.74 mg/mLto 177.69 mg/mL, however, the hydrodynamic diameter was found toincrease.

HSA showed a general tendency of increasing hydrodynamic diameters (peakmonomer) with increasing protein concentration which is in-line withunderlying theoretical principles. The D_(h) decrease from 9.88 mg/mL to22.89 mg/mL is caused by a change in the measurement SOP (switching fromassumed viscosity of 1.1 mPas to an assumed viscosity of 1.9 mPas).

Numerical data describing the above is provided in Appendix A.

Example 9: J695: Impact of Excipients of the Hydrodynamic Diameter

Having found the surprising result that proteins can be dissolved inhigh concentrations in pure water, the impact of ionizable andnon-ionizable excipients typically used in parenteral formulations onthe hydrodynamic diameter was evaluated. J695 was used as a modelprotein.

Table 15 shows that the solution osmolality is directly proportional tothe concentration of sodium chloride. The osmolality in the proteinsolution rises along with the NaCl concentration (an almost linearcorrelation). Interestingly, the hydrodynamic diameter of J695 proteinincreased with increasing salt concentration. NaCl is an ionic excipientand dissociated into positively charged sodium ions and negativelycharged chloride ions which might adsorb at the surface of the protein.Without salt being present, the hydrodynamic diameter of J695 wasdramatically lower than what normally is expected for J695 (usuallyvalues around 10 nm are determined).

As illustrated in Table 15, the osmolality increased linearly with anincrease in concentration of mannitol in the protein solution. Incontrast, the hydrodynamic diameter did not show a dependence onmannitol concentration. Mannitol is a non-ionic sugar alcohol/polyol.Mannitol or polyols are used as stabilizers during parenteralformulation development and in final formulations. Mannitol stabilizesthe protein by preferential exclusion. As other osmolytes, mannitol ispreferentially excluded from the surface of the protein and it isoutside of the hydrate shell of the protein. Thus the folded state ofthe protein is stabilized because the unfolded state, which has a largersurface, becomes thermodynamically less favorable (Foster, T. M.,Thermal instability of low molecular weight urokinase during heattreatment. III. Effect of salts, sugars and Tween 80, 134 InternationalJournal of Pharmaceutics 193 (1996); Singh, S. and Singh, J., Effect ofpolyols on the conformational stability and biological activity of amodel protein lysozyme, 4 AAPS PharmSciTech, Article 42 (2003)).However, it is interesting that the osmolality can be adjusted basicallyas desired—which would be an important feature of the protein findingsdescribed herein—without impacting the D_(h) of the protein. Thesefindings may be useful in high-concentration protein formulation, whereviscosity related manufacturing and dosing issues may be present, as theosmolality adjustment with mannitol is not mirrored by an increase inprotein D_(h) (meaning viscosity is expected to remain constant).

TABLE 15 Impact of Excipients on J695 Osmolality and Z-Average NaClconcentration osmolality z-average [mg/ml] [mosmol/kg] [nm] 0 16 4.19 292 12.2 4 158 16.2 6 230 17 mannitol concentration osmolality z-average[mg/ml] [mosmol/kg] [nm] 0 16 4.19 2 148 5.49 4 276 3.22 6 432 3.54

Example 10: Analysis of High Protein Formulations with Size ExclusionChromatography (SEC)

For the SEC analysis, samples of Adalimumab, J695 and HSA were dilutedto 2 mg/mL before injection. The injection volume for Adalimumab was 20μL. For J695 and HSA, a 10 μL injection volume was used.

10.1: SEC Analysis of Adalimumab

The amount of monomer of Adalimumab tended to slightly decrease from99.4% to 98.8% while concentrating from 9.35 mg/mL to 206.63 mg/mL. Thatdecrease of monomer is associated with an increase in the amount ofaggregate in Adalimumab solution from 0.4% to 1.1% while concentratingfrom 23.27 mg/mL to 206.62 mg/mL, respectively. The amount of fragmentremained constant at 0.1%, independent of protein concentration (seetable in Appendix B). Thus, Adalimumab was stable in WFI.

Overall, the increase in protein aggregation with increasing proteinconcentration is deemed only minor. A similar trend in monomer decreasewould be expected when either the protein was formulated in a buffersystem and when additionally surfactants are added. Adalimumab proteinappears to be surprisingly stable when formulated in pure water.

10.2: SEC Analysis of J695

The amount of J695 monomer slightly decreased from 99.4% to 98.6% withincreasing protein concentration from 9.99 mg/mL to 217.53 mg/mL. Thedecrease of monomer was associated with an increase in aggregate fromabout 0.4% to about 1.2% with increasing protein concentration from 9.99mg/mL to 217.53 mg/mL. Independent from protein concentration, theamount of fragment was almost constant with 0.17% to 0.23% withincreasing protein concentration from 9.99 mg/mL to 217.53 mg/mL.

Overall, the increase in protein aggregation with increasing proteinconcentration was deemed only minor. A similar trend in monomer decreasewould be expected when the protein is formulated in buffer systems andwhen additional surfactants are added. Thus, J695 protein appears to besurprisingly stable when formulated in pure water.

10.3: SEC Analysis of HSA

The amount of monomer HSA decreased from 95.9% to 92.75% whileconcentrating from 9.88 mg/ml to 112.74 mg/mL. For the sample with177.69 mg/mL, an increase in monomer up to 94.5% was determined. Thedecrease of the amount of monomer goes along with an increase in proteinaggregate from 4.1% to 7.25% while concentrating from 9.88 mg/mL to112.74 mg/mL. Thus, HSA protein also appears to be stable whenformulated in pure water.

Numerical data describing the above-mentioned SEC experiments isprovided in Appendix B.

Example 11: Analysis of High ProteinFormulations—Ion-Exchange-Chromatography (IEC)

For the IEC analysis the samples of Adalimumab, J695 and HSA werediluted to 1 mg/mL before injection. The injection volume for allproteins was 100 μL.

11.1: IEC Analysis of Adalimumab

As shown in FIG. 9, Adalimumab was stable in WFI. FIG. 9 shows a slighttrending which may be interpreted as indicating that the sum of lysinevariants (lysine 0, 1 and 2) decreases with an increase concentration ofAdalimumab in WFI. Overall, however, the percentage of lysine variantsvaried less than 0.25%.

11.2: IEC Analysis of J695

FIG. 10 shows that the sum of the J695 peaks 1 to 7 is slightlydecreasing with increasing J695 concentration. With the decrease in peak1-7 the sum of acidic and basic peaks is slightly increasing, with theincrease in the acidic peaks being a little more pronounced (see FIGS.11 and 12). The sum of acidic peaks slightly increases fromapproximately 10.2% to 10.6% and the sum of basic peaks from 0.52% to0.59%, respectively.

Overall, it can be stated that no major instability effects orinsolubility effects of J695 formulations in pure water were observedvia IEC.

Numerical data describing the above IEC experiments is provided inAppendix C.

Summary of Findings in Examples 2-11

It was initially thought that transferring proteins, such as antibodies,into WFI would likely induce protein precipitation by concentrating theprotein beyond its solubility limit in pure water. The above studiesdemonstrate that proteins, including antibodies, not only can betransferred into pure WFI at lower concentrations without encounteringany precipitation phenomena and solubility limitations, but that,surprisingly, Adalimumab (as well as the other two test proteins) can beconcentrated in pure water to ultra-high concentrations beyond 200 mg/mLusing UF/DF and centrifugation techniques (e.g., TFF equipment, Vivaspindevices). In addition, Adalimumab opalescence was unexpectedly found tobe substantially reduced when the protein was formulated in WFI.Osmolality was monitored to ensure that the Adalimumab buffer medium wascompletely exchanged by pure, salt free water (i.e. WFI). Moreover,freeze-thaw processing was performed during sample preparation foranalysis, and virtually no instability phenomena were observed with SECand IEC analysis.

The approach of formulating proteins, e.g., Adalimumab, at highconcentrations in WFI revealed the potential to reduce viscosityphenomena, which often impedes straightforward Drug Product developmentat high protein concentrations.

Finally, the hydrodynamic diameter (determined via photon correlationspectroscopy, PCS) of Adalimumab was found to be notably lower in WFIthan in commercial buffer (indicative of lower viscosity proneness).

Overall, it was concluded that the findings of antibodies and theglobular model protein HSA being soluble in pure water in ultra-highconcentrations have potential to provide new insight into fundamentalprotein regimes and to potentially offer new approaches in protein drugformulation and manufacturing, for instance by:

-   -   reducing opalescence of high-concentrated protein formulations    -   reducing viscosity of high-concentrated protein formulations    -   enabling to adjust osmolality as desired in protein-WFI        solutions by adding non-ionic excipients such as mannitol        without changing features such as viscosity and non-opalescence        (it was demonstrated for J695 that hydrodynamic diameter and        opalescence did not change when mannitol was added, but        increased dramatically when NaCl was added)    -   providing a new paradigm in Drug Substance formulation and        processing, as it was demonstrated that proteins can be        subjected to operations such as DF/UF for concentrating the        protein in WFI to ultra-high concentrations and to freeze and        thaw without substantial stability implications. Given the        background that it is well-known that during DF/UF the        composition of protein formulations, especially during        processing to high concentrations, necessarily changes        (Stoner, M. et al., Protein-solute interactions affect the        outcome of ultrafiltration/diafiltration operations, 93 J.        Pharm. Sci. 2332 (2004)), these new findings could beneficially        be applied by either adjusting the Drug Substance concentration        by DF/UF of the protein in pure water, and add excipients at        high DS concentrations subsequently (by this avoiding the risk        of DS formulation changes during process unit operations).        Alternatively, the excipients could be added to Drug Substance        during final Drug Product fill-finishing.

Example 12: Preparation of Adalimumab in Water Formulation

The following example illustrates the scaling up the DF/UF proceduresresulting in large scale production of adalimumab in water.

12.1: Evaluation of Process Parameters

Dialysis process evaluation studies were performed on a laboratory scaleto define suitable parameters for the dialysis of Bulk Adalimumab DrugSolution formulated in a phosphate/citrate buffer system containingother excipients, e.g., mannitol and sodium chloride (FIGS. 13 and 14).

Conductivity measurements may be taken with any commercially availableconductivity meter suitable for conductivity analysis in proteinsolutions, e.g. conductivity meter Model SevenMulti, with expansioncapacity for broad pH range (Mettler Toledo, Schwerzenbach,Switzerland). The instrument is operated according to the manufacturersinstructions (e.g., if the conductivity sensor is changed in the MettlerToledo instrument, calibration must be performed again, as each sensorhas a different cell constant; refer to Operating Instructions of ModelSevenMulti conductivity meter). If the instructions are followed,conductivity measurements can be taken by directly immersing themeasuring probe into the sample solution.

FIG. 13 shows the efficiency of the dialysis procedure, in terms of thereduction of components responsible for the osmolality and theconductivity of the formulation containing adalimumab at 74 mg/ml. Aftera reduction of the solutes in the antibody solution by a factor of 100,osmolality and conductivity measurements largely stabilized at levelsfar below the original measurements of these parameters from thecommercial formulation.

FIG. 14 shows the stability of pH in dialyzed Adalimumab bulk solutions.pH levels before and after dialysis against deionized water(1:1,000,000) are shown for Adalimumab solutions with a range ofdifferent initial pH readings. pH levels remained nearly the same in theretentate before and after dialysis.

12.2: Production of High-Concentrated Adalimumab in Water Bulk DrugSolution

In a first step, the formulated bulk drug solution (Phosphate/CitrateBuffer system containing other excipients, e.g., Mannitol and SodiumChloride) was up-concentrated by Ultrafiltration/Diafiltration to aconcentration of approximately 100 mg/ml (12 L scale, Millipore Pellicon2 Mini Bio-A MWCO 10 k columns). In a second step, the up-concentratedsolution was dialyzed against deionized water (SpectraPor7 MWCO10 k,dilution factor 1:100,000). As a third step the dialyzed solution wasup-concentrated by Ultrafiltration/Diafiltration to a concentration ofapproximately 250 mg/ml using Millipore Pellicon 2 Mini Bio-A MWCO 10 kcolumns.

Table 16a shows the results of analysis of highly concentratedAdalimumab in water (DF/UF processed) bulk drug solutions after Step 3of the procedure.

TABLE 16a Osmolality and Conductivity Data for DF/UF Bulk-ProcessedAdalimumab osmolality conductivity density Adalimumab mosm/kg mS/cm pHg/cm3 cone mg/ml 62 0.95 5.28 1.0764 277.812.3: Freeze/Thaw (F/T) Procedure Simulating Manufacturing Conditions

Freezing was conducted using an ultra-low temperature freezer (RevcoUltima II, 20 cu·ft.) with a manufacturing scale load of 47 kg of liquidto be frozen at a temperature below −50° C., typically −70° C. to −80°C. The liquid was packaged in individual bottles of 1.6 kg fill weight(e.g., Nalgene 2 L PETG square media). Freezing was completed after 48hours. Thawing was conducted in a circulating water bath (e.g.,Lindergh/Blue) with a manufacturing scale load of 24 kg at a temperaturebetween 20° C. and 40° C., typically 30° C., until the material wascompletely thawed.

12.4: Bottle Mapping During Freeze and Thaw

Individual horizontal solution layers in the bottle volume were isolatedand analyzed. At protein concentrations of 250 mg/ml and 200 mg/ml, onlyminimal gradient formation was detected in the Adalimumab watersolution, as seen in FIGS. 15 through 19. Freezing and thawing offormulated Adalimumab solutions (solutions with a Phosphate/CitrateBuffer system containing other excipients, e.g., Mannitol and SodiumChloride) at 250 mg/ml and 200 mg/ml, however, led to the formation ofprecipitate on the bottom of the bottle.

12.5: Gradient Formation in Commercial and Low-Ionic Formulations ofAdalimumab

The formation of gradients by freeze thaw procedures in commercial andlow-ionic (water) formulations of Adalimumab was compared. Table 16bshows the results of visual inspection of commercial Adalimumabsolutions of various concentrations after a f/t step. The formation ofprecipitates indicates that instability was created in the solution bythe f/t procedure. Above 100 mg/ml, significant precipitate formationwas observed. Table 17 shows the analytical data of two 50 mg/mlsolutions and one 100 mg/ml low-ionic formulation before the freeze-thawexperiment.

TABLE 16b Observed Precipitation in Commercial Adalimumab Solutionsafter F/T 250 mg/ml 220 mg/ml 200 mg/ml 150 mg/ml 120 mg/ml 100 mg/ml 60mg/ml Commercial Precipitate Precipitate Precipitate Precipitate PartialClear Clear freezing Precipitate process in ultra low temp freezer: −70°C./23° C.

TABLE 17 Solution Analytical Data before Freeze-Thaw Osmolality ProteinDensity (mOsmol/ conc formulation pH (g/cm³) kg) (mg/mL) E167 130 01 CLlow-ionic 5.18 1.0121 5 49.3 50 mg/mL in water E167 140 01 CL Commercial5.20 1.0224 280 48.7 50 mg/mL in formulation buffer low-ionic 5.321.0262 12 99.8 100 mg/mL in waterAbout 1600 mL (50 mg/ml solutions) or 800 ml (100 mg/ml solution) ofeach formulation were placed into PETG bottles and subjected to aconventional freeze (−80° C.) thaw (23° C., water bath) procedures.Samples were then pulled from top, center and bottom of the PETG bottlesand analyzed for pH, density, osmolality, and protein concentration.Analysis results are shown in Table 18.

TABLE 18 Analysis of Bottle-Mapped Layers from Frozen/Thawed Solutionsprotein osmolality content density mOsmol/ (volumetric) sample pH g/cm3kg mg/mL 50 mg/ml in water top 5.20 1.0119 6 48.72 middle 5.19 1.0120 849.35 bottom 5.17 1.0120 6 49.76 commercial formulation top 5.16 1.0165236 37.9 middle 5.13 1.0221 306 45.58 bottom 5.12 1.0257 368 55.48 100mg/ml in water top 5.29 1.0259 13 98.7 middle 5.3 1.0262 16 99.9 bottom5.28 1.0262 14 101.2

The commercial formulation of Adalimumab revealed significant gradientsupon freeze/thaw with regard to density (indicatingheterogeneities/gradients of protein and excipients), osmolality(indicating excipient gradients), and protein content. In contrast, nogradients were found in the 50 mg/ml low-ionic Adalimumab formulationupon freeze/thaw.

At higher protein concentrations, gradient formation may sometimes beexpected to become worse. However, no gradients were found in the 100mg/ml low-ionic Adalimumab formulation upon freeze/thaw with regard topH, density, osmolality and protein concentration.

Example 13: Stability of J695 after DF/UF

The following example provides data on the stability of J695 after DF/UFprocessing in accordance with the methods of the invention.

Protein samples from J695 in normal DS buffer were analyzed, eitherafter pH adjustment or after diafiltration. pH was adjusted to pH 4.4with 0.1 M phosphoric acid, protein concentration 112 mg/mL. Forconcentrated samples in WFI, protein samples were diafiltered (DF/UF)against water for approximately 1.5 days at ambient temperature, using aTFF equipped with a 30 kDa RC membrane. The protein concentration afterDF/UF was determined approx. 192 mg/mL. pH 4.7.

13.1: Size Exclusion Analysis (SEC) Experimental Procedures

A size exclusion method was developed for the purity assessment of J695.Size exclusion chromatography (SEC) separates macromolecules accordingto molecular weight. The resin acts as a sieving agent, retainingsmaller molecules in the pores of the resin and allowing largermolecules to pass through the column. Retention time and resolution arefunctions of the pore size of the resin selected.

Each sample was diluted to 2.5 mg/mL with purified water (Milli-Q) basedon the stated concentration. 50 μg of each sample was injected onto thecolumn in duplicate. A Tosoh Bioscience G3000swxl, 7.8 mm×30 cm, 5 μm(Cat #08541) SEC column is used for separation. For Buffer A, 211 mMNa₂SO₄/92 mM Na₂HPO₄, pH 7.0 was used. Detection was performed at 280 nmand 214 nm. The column was kept at room temperature with a flow rate of0.3 mL/min.

This chromatography utilized an isocratic gradient with a 100% mobilephase A solvent for 50 minutes duration.

13.2: SEC Data

Table 19 describes data from size exclusion chromatography experiments.

TABLE 19 SEC Analysis Data for J695 Reference Standard, DS andPost-DF/UF (in water) ABT-874 load HM Monom Frag BF Ref Std 50 u 0.4997.9 1.28 0.26 BF Ref Std dup 50 u 0.41 98.0 1.29 0.27 BF Ref std avg0.45 98.0 1.29 0.27 std 0.06 0.05 0.01 0.01 % RS 12.5 0.05 0.55 2.67 DSin Buffer pH 4.4 50 u 0.42 98.3 1.04 0.16 DS in Buffer pH 4.4 dup 50 u0.40 98.4 1.01 0.13 DS in Buffer pH 4.4 avg 0.41 98.4 1.03 0.15 std 0.010.06 0.02 0.02 % RS 3.45 0.06 2.07 14.6 DS UF/DF in Water pH 4.7 50 u0.69 98.1 1.04 0.14 UF/DF in Water pH 4.7 dup 50 u 0.69 98.0 1.07 0.16DS UF/DF in Water pH 4.7 avg 0.69 98.1 1.06 0.15 std 0.00 0.04 0.02 0.01% RS 0.00 0.04 2.01 9.43

The data in Table 19 shows that the commercial formulation of J695 (DSPFS, pH=4.4) has comparable levels of fragments and aggregate as theJ695 reference standard. There was a difference noted in the aggregateamount between the commercial formulation J695 control and the J695 thathad undergone DF/UF, in water (DS in H2O, pH=4.7, 192 mg/ml): anincrease from 0.4% to 0.7% in aggregation was seen. This was not asignificant increase and may be due to time spent at room temperatureduring the UF/DF. There is no change to the fragments.

13.4: IEC (WCX-10) Experimental Procedure

A cation exchange method was developed for the assessment of theheterogeneity of J695 using the Dionex WCX-10 column. Generally, cationexchange chromatography separates protein isoforms according to theapparent pI and the surface charge interaction with the resin. Theprotein of interest is bound to the column under specific low saltstarting conditions and is eluted from the column by increasing the saltconcentration through a gradient. Proteins with lower apparent pI bindless tightly to a cation exchange column and are the first to elute andproteins with a higher apparent pI bind tighter and are the last toelute.

Cation exchange chromatography using WCX-10 was used in quality controlas a lot release assay. The assay conditions were modified to improveseparation of known J695 isoforms.

The sample was diluted to 1.0 mg/mL with purified water (Milli-Q).Reference standard was run in triplicate as a comparison and was dilutedto 1 mg/ml in purified water (Milli-Q).

Dionex Propac WCX-10 columns (p/n 054993), along with correspondingguard columns (p/n 054994), were used for separation. Buffers used inthe procedure included Buffer A (10 mM Na₂HPO₄, pH=6.0) and Buffer B (10mM Na₂HPO₄, 500 mM NaCl, pH=6.0). Column temperature was maintained at35° C. and column flow rate was 1 mL/min. Injection volumes were 100 μlfor a 100 μg load and detection was performed at 280 nm. Buffergradients over the course of the chromatographic separation are providedin Table 20.

TABLE 20 Buffer Gradients used in IEC Analysis of J695 Time (min) % MPA% MPB 0 75 25 3 60 40 33 40 60 36 0 100 41 0 100 43 75 25 48 75 2513.5: IEC Data

Table 21 provides results from experiments comparing the J695 ReferenceStandard to J695 in commercial buffer (DS pH=4.4), as well as acomparison of the commercial buffer formulation to J695 after DF/UF(DF/UF H2O, pH=4.7).

TABLE 21 IEC Data for J695 Reference Standard, Commercial Formulation(DS) and after DF/UF (in water) 0 glu 0 glu 1 glu 1 glu 1 glu 2 glu 2glu (1) (2 + 2a) (3) (4) (5) + (5a) (6) (7) acidic basic ref std 43.777.55 8.00 21.87 4.28 4.82 3.75 4.05 1.92 ref std dup 43.49 7.49 7.9821.70 4.26 4.81 3.75 4.61 1.90 ref std dup 43.44 7.49 8.00 21.65 4.244.81 3.74 4.75 1.89 ref std avg 43.57 7.51 7.99 21.74 4.26 4.81 3.754.47 1.90 SD 0.20 0.04 0.01 0.12 0.01 0.01 0.00 0.40 0.01 % RSD 0.450.56 0.18 0.55 0.33 0.15 0.00 8.86 0.74 ABT-874 DS pH = 4.4 35.65 14.747.26 18.06 6.76 5.32 3.98 5.55 2.70 ABT-874 DS pH = 4.4 Dup 35.82 14.737.29 18.14 6.82 5.39 4.06 4.79 2.95 ABT-874 DS pH = 4.4 avg 35.74 14.747.28 18.10 6.79 5.36 4.02 5.17 2.83 SD 0.12 0.01 0.02 0.06 0.04 0.050.06 0.54 0.18 % RSD 0.34 0.05 0.29 0.31 0.62 0.92 1.41 10.39 6.26ABT-874 DF/UF H2O 36.57 14.51 7.26 18.09 6.57 5.22 3.91 5.28 2.61 pH =4.7 ABT-874 DF/UF H2O 36.60 14.43 7.25 18.02 6.66 5.18 4.00 5.31 2.56 pH= 4.7 Dup ABT-874 DF/UF H2O 36.59 14.47 7.26 18.06 6.62 5.20 3.96 5.302.59 pH = 4.7 avg SD 0.02 0.06 0.01 0.05 0.06 0.03 0.06 0.02 0.04 % RSD0.06 0.39 0.10 0.27 0.96 0.54 1.61 0.40 1.3713.6: IEC Analysis Conclusions

There were some differences noted between the J695 Reference Standardand the commercial formulation (DS, pH 4.4). These differences werenoted in the initial run of the DS engineering run sample and areattributed to differences in the manufacturing processes between the3000 L and 6000 L campaigns. There were no notable differences betweenthe DS, pH 4.4 control and the J695 in H₂O pH=4.7, 192 mg/ml sample.

Example 14: Stability of Adalimumab after DF/UF and Long-Term Storage at2-8° C.

The following example provides data showing the stability of Adalimumabin an aqueous formulation in accordance with the methods of theinvention, after 22.5 months storage at 2-8° C.

Adalimumab samples for SEC and WCX-10 analysis were diafiltered againstwater and concentrated to about 177 mg/mL. Samples were stored andanalyzed at various time points for stability.

Standard Adalimumab solution (DS, pH approx. 5.2) in commercial Humirabuffer was used as a starting material for generating a concentratedsolution in water. Protein solution samples were diafiltered (DF/UF)against water for approximately 1.5 days at ambient temperature, using aTFF equipped with a 30 kDa RC membrane. Protein concentration afterDF/UF was determined approximately 177 mg/mL, pH 5.2. The sample wasstored at 2-8° C. for 22.5 months before analysis.

14.1: SEC Experimental Procedure

A size exclusion method was previously developed to check for thepresence of antibody fragments and aggregates. Size exclusionchromatography (SEC) separates macromolecules according to molecularweight. The resin acts as a sieving agent, retaining smaller moleculesin the pores of the resin and allowing larger molecules to pass throughthe column. Retention time and resolution are functions of the pore sizeof the resin selected.

Each sample was diluted to 1.0 mg/mL with milli Q water and 50 μg ofeach sample was injected onto the column. For SE-HPLC, a Sephadex 200column (Pharmacia cat #175175-01, S/N 0504057) or a TSK gel G3000SW (cat#08541; for analysis of 22.5 month samples) were used. The mobile phaseof the column comprised 20 mM Sodium phosphate and 150 mM Sodiumchloride, pH 7.5. Detection was performed at 280 nm and 214 nm. Columnswere kept at ambient temperature and the flow rate was 0.5 mL/min(Sephadex column), or 0.3 mL/min (TSK column).

14.2: SEC Data

FIG. 20 and Table 22 contain results of the analysis of a low-ionicAdalimumab solution stored as a liquid at 2-8° C. for 8.5 monthscompared to the same solution stored at −80° C. Table 23 containsanalysis data for a low-ionic Adalimumab solution stored at 2-8° C. for22.5 months compared to a reference standard sample of Adalimumab.

TABLE 22 SEC Analysis Data Comparing Adalimumab from Frozen Storageversus Adalimumab from Long-Term Refrigerated Storage % % % Sample LoadHMW Monomer LMW DF/UF against water, 50 μg 0.1 99.6 0.3 177 mg/mL, 4.5months at −80° C. DF/UF against water, 50 μg 0.2 99.5 0.3 177 mg/mL, 9months at 2-8° C.

TABLE 23 SEC Analysis Data Comparing Adalimumab Reference Standardagainst Adalimumab from Long-Term Refrigerated Storage % % % Sample LoadHMW Monomer LMW Reference Std. Adalimumab 50 μg 0.31 98.85 0.84 DF/UFagainst water, 177 mg/mL, 50 μg 1.42 97.59 0.98 22.5 months at 2-8° C.As can be seen in Table 22, SEC analysis revealed that adalimumab inwater was stable even after 9 months at 2-8° C. or for 4.5 months at−80° C., as the percent aggregate (% HMW) and percent fragment (% LMW)were minimal over time.14.3: SEC Analysis Conclusions

After 8.5 months storage at 2-8° C., the Adalimumab solution (DF/UFagainst water) revealed a small fraction of high molecular weight (HMW)species (0.2%) and a small fraction of fragment (0.3%). Storage for 4.5months at −80° C. and subsequent thaw (water bath, 23° C.) did notimpact Adalimumab stability (0.1% aggregate, 0.3% fragment).

Analysis of a sample stored for 22.5 months at 2-8° C. also showscomparable fragment content to Adalimumab reference standard (Table 23).However, the aggregate levels detected in the 22.5 month stabilitysample (1.66%) are somewhat higher than aggregate levels detected in thereference standard.

It is known that self-association of antibodies is highly dependent onthe antibody concentration, i.e. the formation of non-covalent aggregateand associate complexes is most pronounced at high proteinconcentration. This self-association is reversible, and dilution withbuffer solution results in reduced self-association tendencies (Liu, J.et al., 94 Journal of Pharmaceutical Sciences 1928 (2004)).

Thus, it is likely that differences in sample preparations and differentlag-times between Adalimumab solution dilution (from 177 mg/mL to 1mg/mL) and subsequent sample analysis by SEC are the reason for thedifferences in aggregate content of the 8.5 month and the 9 monthstability samples.

14.4: IEC Experimental Procedure

A cation exchange method was developed for the assessment of antibodycharge heterogeneity using the Dionex WCX-10 column. Cation exchangechromatography separates protein isoforms according to the apparent pIand the surface charge interaction with the resin. The protein ofinterest is bound to the column under specific low salt startingconditions and is eluted from the column by increasing the saltconcentration through a gradient. Proteins with lower apparent pI bindless tightly to a cation exchange column and are the first to elute andproteins with a higher apparent pI bind tighter and are the last toelute.

Before the procedure, samples were diluted to 1.0 mg/mL with milli Qwater. Dionex Propac WCX-10 columns (p/n 054993), along with acorresponding guard columns (p/n 05499), were used for separation. Twomobile phase buffers were prepared, 10 mM Sodium phosphate, pH 7.5(Buffer A) and 10 mM Sodium phosphate, 500 mM Sodium chloride, pH 5.5(Buffer B). Columns were kept at ambient temperature and the flow ratewas 1.0 mL/min. Injection volumes were 100 μl for a 100 μg load anddetection was performed at 280 nm. Buffer gradients over the course ofthe chromatographic separation are provided in Table 24.

TABLE 24 Buffer Gradients used in WC Analysis of Adalimumab Time (min) %MPA % MPB 0.05 94 6 20 84 16 22 0 100 26 0 100 28 94 6 34 94 6 35 94 614.5: Ion Exchange Data

Table 25 shows the ion exchange chromatographic data for the Adalimumabreference standard, commercial formulation (150 mg/ml) and post-DF/UFlow-ionic solution before storage. Table 26 shows data for the referencestandard compared to the low-ionic solution after 22.5 months of storageat 2-8° C.

TABLE 25 IEC Analysis Data of Adalimumab Reference Standard,DS/Commercial Formulation and After DF/UF (in water) % Acidic % Acidic %0 % 1 % 2 Sample Name Region 1 Region 2 Lys Lys Lys Adalimumab Ref. Std.2.69 11.66 60.77 19.42 5.40 Adalimumab DS 150 mg/ml 2.51 11.38 62.0519.14 4.83 Adalimumab diafiltered 2.26 11.81 61.97 18.51 4.73 againstwater, 177 mg/ml

TABLE 26 IEC Analysis Data Comparing Reference Standard to DF/UF Samplefrom Long-Term Refrigerated Storage % Acidic % Acidic % 0 % 1 % 2 SampleName Region 1 Region 2 Lys Lys Lys Adalimumab Ref. Std. 2.1 10.9 63.818.4 4.6 Adalimumab DF/UF against 2.7 13.4 62 16.7 4.1 water, 177 mg/mL,22.5 months at 2-8° C.14.6: Ion Exchange Analysis Conclusions

For the T0 samples, data show no significant difference in thepercentage of acidic region 1, 2, 0 Lys, 1 Lys, or 2 Lys (i.e., chargeheterogeneity) between reference standard Adalimumab, commercialformulation Adalimumab (used as DS to formulate Adalimumab into water byDF/UF), and Adalimumab diafiltered against water and concentrated to 177mg/ml (Table 25).

Also, after 22.5 months storage of the 177 mg/mL Adalimumab sample inwater, only slight differences in 0 Lys, 1 Lys and 2 Lys fractions canbe seen when compared to the Adalimumab reference standard. In summary,no significant chemical instability tendencies are observed whenAdalimumab is formulated into water by DF/UF processing and stored for22.5 months at 2-8° C. at a concentration of 177 mg/mL.

Example 15: Freeze/Thaw Stability of Low-Ionic 1D4.7 Solution

1D4.7 protein (an immunoglobulin G1) anti-IL 12/anti-IL 23 wasformulated in water by dialysis (using slide-a-lyzer cassettes, usedaccording to operating instructions of the manufacturer, Pierce,Rockford, Ill.) was demonstrated to be stable during repeatedfreeze/thaw (f/t) processing (−80° C./25° C. water bath) at 2 mg/mLconcentration, pH 6. Data were compared with routine formulations (2mg/mL, pH 6), and it was found that the stability of 1D4.7 formulated inwater exceeded the stability of 1D4.7 formulated in routinely screenedbuffer systems (e.g. 20 mM histidine, 20 mM glycine, 10 mM phosphate, 10mM citrate) and even exceeded the stability of 1D4.7 formulations basedon universal buffer (10 mM phosphate, 10 mM citrate) with a variety ofexcipients that are commonly used in protein formulation, e.g. 10 mg/mLmannitol, 10 mg/mL sorbitol, 10 mg/mL sucrose, 0.01% polysorbate 80, 20mM NaCl.

SEC, DLS and particle counting was performed to monitor proteinstability, and particle counting was performed by using a particlecounting system with a 1-200 μm measurement range (e.g. particle counterModel Syringe, Markus Klotz GmbH, Bad Liebenzell, Germany). Experimentdetails are as follows:

-   -   1D4.7 formulated in water compared with formulations listed        above    -   4 freeze/thaw cycles applied    -   30 mL PETG repository, about 25 mL fill, 2 mg/mL, pH 6    -   sampling at T0, T1 (i.e. after one f/t step), T2, T3, and T4    -   analytics: visual inspection, SEC, DLS, subvisible particle        measurement

FIG. 21 shows 1D4.7 stability during repeated f/t cycling (−80° C./25°C.), mirrored by formation of subvisible particles >1 μm. 1D4.7 wasformulated in universal buffer (10 mM citrate, 10 mM phosphate) and thenthe following excipient variations were tested: sorbitol (10 mg/mL),mannitol (10 mg/mL), sucrose (10 mg/mL), NaCl (100 mM), and polysorbate80 (0.01%). 1D4.7 was also formulated in water (by dialysis) with noexcipients added at all. Water for injection was also subjected to f/tcycling and subvisible particle testing to evaluate a potential impactof material handling, f/t, and sample pull on particle load.

The stability of 1D4.7 formulated in water upon f/t exceeded thestability of 1D4.7 solutions formulated with excipients typically usedin protein formulations. Mannitol, sucrose, and sorbitol are known toact as lyoprotectant and/or cryoprotectant, and polysorbate 80 is anon-ionic excipient prevalently known to increase physical stability ofproteins upon exposure to hydrophobic-hydrophilic interfaces such asair-water and ice-water, respectively. Thus, 1D4.7 solutions formulatedin water appeared to be stable when analyzed with other methodologiesapplied (e.g. SEC, visual inspection, etc.).

Example 16: Freeze/Thaw Stability of Low-Ionic 13C5.5 Antibody Solution

13C5.5 anti IL-13 protein formulated in water was demonstrated to bestable during repeated freeze/thaw processing (−80° C./25° C. waterbath) at 2 mg/mL concentration, pH 6. Data were compared with routineformulations (2 mg/mL, pH 6), and it was found that the stability of13C5.5 formulated in water exceeded the stability of 13C5.5 formulatedin routinely screened buffer systems (e.g. 20 mM histidine, 20 mMglycine, 10 mM phosphate, 10 mM citrate) and even exceeded the stabilityof 13C5.5 formulations based on universal buffer (10 mM phosphate, 10 mMcitrate) with a variety of excipients that are commonly used in proteinformulation (e.g. 10 mg/mL mannitol, 10 mg/mL sorbitol, 10 mg/mLsucrose, 0.01% polysorbate 80, 20 mM NaCl, 200 mM NaCl).

Sample preparation, experiment processing, sample pull and sampleanalysis was performed in the same way as outlined in Example 15 for1D4.7.

-   -   13C5.5 formulated in water compared with formulations listed        above    -   4 freeze/thaw cycles applied    -   30 mL PETG repository    -   2 mg/mL, pH 6    -   sampling at T0, T1, T2, T3, and T4    -   analytics: visual inspection, SEC, DLS, subvisible particle        measurement

FIG. 22 shows 13C5.5 stability during repeated f/t cycling (−80° C./25°C.), mirrored by formation of subvisible particles >10 μm. 13C5.5 wasformulated in either 10 mM phosphate buffer, 10 mM citrate buffer, 20 mMglycine buffer, and 20 mM histidine buffer. 13C5.5 was also formulatedin water (by dialysis) with no excipients added at all. Water forinjection was also subjected to f/t cycling and subvisible particletesting to evaluate a potential impact of material handling, f/t, andsample pull on particle load (blank).

The stability of 13C5.5 formulated in water upon f/t exceeded thestability of 13C5.5 solutions formulated in buffers typically used inprotein formulations. No instabilities of 13C5.5 solutions formulated inwater have been observed with other analytical methodologies applied(e.g. SEC, visual inspection, etc.)

FIG. 23 shows 13C5.5 stability during repeated f/t cycling (−80° C./25°C.), mirrored by formation of subvisible particles >1 μm. 13C5.5 wasformulated in universal buffer (10 mM citrate, 10 mM phosphate) and thenthe following excipient variations were tested: sorbitol (10 mg/mL),mannitol (10 mg/mL), sucrose (10 mg/mL), NaCl (200 mM), NaCl (20 mM) andpolysorbate 80 (0.01%). 13C5.5 was also formulated in water (bydialysis) with no excipients added at all for comparison (pure water).Water for injection was also subjected to f/t cycling and subvisibleparticle testing to evaluate a potential impact of material handling,f/t, and sample pull on particle load.

The stability of 13C5.5 formulated in water upon f/t exceeded thestability of 13C5.5 solutions formulated with excipients typically usedin protein formulations. Mannitol, sucrose, and sorbitol are known toact as lyoprotectant and/or cryoprotectant, and polysorbate 80 is anon-ionic excipient prevalently known to increase physical stability ofproteins upon exposure to hydrophobic-hydrophilic interfaces such asair-water and ice-water, respectively.

No instabilities of 13C5.5 solutions formulated in water have beenobserved with other analytical methodologies applied, (e.g. SEC, visualinspection, etc.).

DLS analysis of 13C5.5 solutions after f/t procedures was performed asdescribed above. An 13C5.5 solution with 0.01% Tween-80 containedsignificant high molecular weight (HMW) aggregate forms after only 1 f/tstep, whereas 13C5.5 in water contained no HMW aggregate forms, evenafter 3 f/t steps.

Example 17: Impact of Solution pH on Adalimumab in WFI

The following experiments were performed to determine the impact ofsolution pH on physico-chemical characteristics of highly concentratedAdalimumab formulated in WFI. The following concentrations were tested:2 mg/mL, 50 mg/mL, 100 mg/mL, 150 mg/mL, 200 mg/mL, and 250 mg/mL.

Materials

-   -   Adalimumab Drug Substance (DS), commercial material    -   25° C. water bath (circulating) used for thawing    -   Diafiltration equipment: Sartorius Sartocon Slice, membrane: PES        50 kD, 1000 cm²    -   Diafiltration equipment: Millipore Labscale™ TFF System,        membrane: PLCTK 30 kD, regenerated Cellulose, size: 50 cm²    -   Eppendorf Centrifuge 5810 R    -   Amicon Ultra-15 repositories for centrifugation, Ultracel-30 k,        Regenerated Cellulose 30,000 MWCO    -   Millex GV 0.22 μm, Millipore for sterile filtration of samples    -   Sample repositories (Eppendorf sample repository 1.5 mL, Roth        cryovials 5 mL, PETG bottle 125 mL)

Analytics:

-   -   pH measurement using Biothrode    -   Density measurement    -   Osmolality measurement    -   UV/VIS spectrophotometer for protein concentration measurement    -   Photon Correlation Spectroscopy (PCS)    -   Viscosity measurement    -   Turbidity measurement    -   Size Exclusion Chromatography (SEC)    -   Fourier transform mid infrared spectroscopy (FT-M-IR)        17.1 Overview of Preparation for DF/UF of Adalimumab Commercial        Formulation

The Adalimumab DS solution (120 mg/mL) was divided into 7 volumeportions which were adjusted to pH3, pH4, pH5, pH6, pH7, pH8, pH9 with0.25N NaOH and 0.25N HCl, respectively. Then the samples were dilutedwith Adalimumab buffer of the respective pH to 100 mg/mL. The solutionsrevealed a slight cloudyness that disappeared after sterile filtration(0.22 μm, PVDF sterile filter). After dilution, the pH value weremonitored again (see Table 27 below).

The following samples of the 100 mg/mL solutions were pulled from eachsolution:

-   -   4 mL for turbidity and subsequent zetapotential measurement    -   1 mL for viscosity measurement (using dropping-ball viscometer)    -   0.15 mL for osmolality measurement    -   2 mL for density measurement    -   0.15 mL for PCS (sample viscosity taken into account for        measurements)    -   1 mL for FT-M-IR    -   2 mL for viscosity and static light scattering measurements

The samples for zetapotential, viscosity and static light scatteringmeasurements were frozen (−80° C.). The remaining volumes of pH 4, pH 5,pH 6, pH 7, and pH 8 solutions were subjected to continuous modediafiltration using water for injection as exchange medium. The sampleswere first frozen at −80° C. Before DF/UF, the samples were thawed at25° C. in a Julabo water bath.

17.2 DF/UF and Concentration Procedures

Adalimumab solutions in commercial formulation, with concentrations of100 mg/ml, with pH levels of 4, 5, 6, 7 and 8, were subject to DF/UFprocessing and further subject to concentration process with UF in acentrifuge. This section describes the processing of the pH 6 Adalimumabsolution as an example. Processing for the other solutions was done in asimilar manner.

The Adalimumab solution (100 mg/mL, pH 6) was thawed in a water bath at25° C. and then homogenized. Then, the solution was subjected todiafiltration using water for injection as exchange medium with TFFequipment M.P. 33.4 by applying the following parameters:

-   -   stirrer: speed 2    -   pump: speed 1    -   pressure up-stream/inlet: 2-2.4 bar    -   pressure down-stream/outlet: 0.6-0.8 bar    -   membrane: regenerated Cellulose, cut off 30 kD    -   continuous mode DF/UF    -   about 6-fold volume exchange applied during DF/UF operation

After applying 6-volume exchange steps, the concentration of Adalimumabwas determined by means of OD280, photometer M.P. 9.7. The osmolality ofpermeate and retentate was checked.

concentration: 125.1 mg/mL

osmolality permeate: 57 mOsmol/kg

osmolality retentate: 12 mOsmol/kg

The Adalimumab solution in water after DF was diluted with water forinjection to 100 mg/mL and sterile filtered. The following samples werepulled from 100 mg/mL solution after the DF/UF process:

-   -   4 mL for turbidity and subsequent zetapotential measurement    -   1 mL for viscosity measurement    -   0.15 mL for osmolality measurement    -   2 mL for density measurement    -   0.15 mL for PCS (viscosity taken into account during        measurement)    -   0.15 mL for SEC    -   pH—measurement    -   1 mL for FT-M-IR    -   2 mL for viscosity and static light scattering measurements

A portion of the 100 mg/mL Adalimumab solution was diluted with waterfor injection to create 50 mg/mL and 2 mg/mL solutions. The followingsamples were pulled from both solutions:

-   -   4 mL for turbidity and subsequent zetapotential measurement    -   2 mL for viscosity measurement    -   0.15 mL for osmolality measurement    -   2 mL for density measurement    -   0.15 mL for PCS (viscosity taken into account)    -   pH—measurement

Adalimumab solutions (pH 6, 100 mg/mL) in water were subjected toconcentration experiments using centrifugation. Centrifugation wasperformed with Eppendorf Centrifuge (5810R M.P. 33.57). Eachcentrifugation step was applied for 15 min. at 4000 rpm. After that,homogenization of sample solution in the centrifuge concentration devicewas performed by gentle upside-down rotation in order to homogenized thesolution and thereby to avoid gel formation in areas immediatelyadjacent to the membrane. Temperature during concentration was 15° C.The centrifugation was performed to about 250 mg/mL. The concentrationwas determined by means of measuring OD280, photometer M.P. 9.7. TheAdalimumab solutions were then diluted to concentrations of 250 mg/mL,200 mg/mL and 150 mg/mL.

The following samples were pulled after the concentration procedure andafter each individual step of dilution. Sample volumes pulled from 250mg/mL and 150 mg/mL solutions were:

-   -   2 mL for viscosity—measurement    -   0.15 mL for PCS (viscosity taken into account)    -   0.15 mL for osmolality measurement    -   0.15 mL for SEC    -   pH—measurement        Sample volumes pulled from the 200 mg/mL solution were:    -   4 mL for turbidity and subsequent zetapotential measurement    -   1 mL for viscosity measurement    -   0.15 mL for osmolality measurement    -   2 mL for density measurement    -   0.15 mL for PCS (viscosity taken into account)    -   0.15 mL for SEC    -   pH—measurement    -   2 mL for analytical work to be performed at ABC (viscosity and        static light scattering measurements)

The concentration processing of Adalimumab solution in water was haltedat approximately 250 mg/mL at each pH value because the viscosity ofAdalimumab solution in water at higher concentrations, and especially atpH values close to the pI (about pH 8.5 for Adalimumab), increaseddramatically (viscosities approaching gel formation).

17.3 Visual Inspection of Adalimumab Solutions

After DF/UF and concentration to 250 mg/mL, the Adalimumab solutions inwater at various pH appeared less opalescent than the Adalimumabsolution in buffer (commercial formulation). All of the Adalimumabsolutions in water appeared as clear solutions at each pH value. None ofthe Adalimumab solutions revealed opalescence after dilution. Overall,during concentration and dilution procedures, no precipitation wasobserved in Adalimumab solutions in water.

17.4 Viscosity

The viscosity measurements were performed taking into account thedensity of pH 5 Adalimumab solutions at each of the respectiveconcentrations. A dropping-ball viscometer was used. Viscosities higherthan 200 mPa*s were measured using capillary viscometer.

FIG. 24 provides an overview of viscosity data of Adalimumab solutionsin water with pH ranging from 4 to 8, at various concentrations (2 mg/mLto 250 mg/mL, in 50 mg/mL concentration steps). There is a clearcorrelation between solution pH, concentration and viscosity. Theviscosity increases with increases of protein concentration, independentof the solution pH. At solution pH values close to the pI of Adalimumab(i.e. pH 7 and pH 8), increases in solution viscosity were mostpronounced, especially at higher protein concentrations (i.e. 200 mg/mL,250 mg/mL).

17.5 Turbidity

As seen in FIG. 25, the same trend was found for turbidity data, (i.e.,the turbidity increased with increasing concentration and withincreasing pH). All samples were sterile filtered (0.22 μm) beforeturbidity measurement.

17.6 Hydrodynamic Diameter (PCS)

The PCS measurements were performed taking into account the viscosityfor each sample, at each concentration and at each pH value. Solutionsat 200 mg/mL and 250 mg/mL were measured but were outside the testingparameters of the Zetasizer nano series (Malvern Instruments) equipment,and consequently the data from these measurements was not analyzed.

The hydrodynamic diameter (Dh) was found to be notably decreased whenAdalimumab was formulated in water (Dh about 2 nm at 50 mg/mL, pH 5) incomparison to Adalimumab formulated into commercial formulation (Dhabout 7 nm). FIG. 26 illustrates the PCS data (also found in Table 39).Corresponding data tables are shown below in part 17.11.

As shown in FIG. 26, for solutions at pH values of 4, 5 and 6, the Dh ofAdalimumab monomer decreased constantly with increased proteinconcentration. In contrast, solutions with pH values closer to the pI ofAdalimumab (i.e., at pH 7 and pH8) showed considerable increases in Dhas concentration increased from 2 mg/mL to 50 mg/mL. As concentrationsrose beyond 50 mg/mL in pH 7 and 8 solutions, however, Dh decreased. Ata concentration of 150 mg/mL, all of the solutions had lower Dh valuesthan the corresponding pH solution at 2 mg/mL. FIG. 27 shows Dh sizedistributions for pH 5 solutions of various concentrations. FIG. 28shows Dh size distributions for five Adalimumab solutions formulated inwater, each having a 100 mg/mL protein concentration and a different pHvalue. FIG. 29 shows data similar to data in FIG. 28, except that thefive Adalimumab solutions were formulated in buffer.

17.7 pH—Measurement

Measurements of solution pH were performed at 100 mg/mL before and afterDF/UF using water (i.e., performed on Adalimumab formulated in bufferand in water, respectively). Table 27 shows the results. The pH valuesstay constant at pH 5, pH 6 and pH 7 before and after DF/UF. Thesolution pH does not change because of a medium change. The pH value atpH 4 slightly increases and at pH 8 slightly decreases after DF/UF usingwater.

TABLE 27 pH values before and after DF/UF with water pH4 pH5 pH6 pH7 pH8Adalimumab 100 4.00 4.99 6.00 7.03 8.00 mg/mL in buffer Adalimumab 1004.29 4.98 5.98 7.02 7.67 mg/mL in water17.8 Osmolality Measurements

During DF/UF of the pH 5 solution samples, solution osmolality wasmeasured after each volume exchange step (i.e., after 100 mL permeate,200 mL permeate, etc.) to check whether a 5-fold volume exchange issufficient to reduce osmolality to values below 15 mOsmol/kg. Table 28shows the results.

TABLE 28 Osmolality change during DF/UF using water, pH 5 solutionVolume exchange step Retentate Permeate in mL mOsmol/kg mOsmol/kg 100 96166 200 28 115 300 29 89 400 12 67 500 15 49

At pH 4, pH 6, pH 7 and pH 8, the osmolality was measured at the end ofthe DF/UF process only. Table 29 shows the osmolality results (inmOsmol/kg units) for each pH.

TABLE 29 Osmolality at various pH values, before and after DF/UF withwater pH4 pH5 pH6 pH7 pH8 Adalimumab 100 287 298 297 286 279 mg/mL inbuffer Adalimumab 100 40 13 11 5 5 mg/mL in water

The osmolality measurements were performed with a freezing pointviscometer.

17.9 Fragmentation (SEC)

The SEC data show a relative pronounced fragmentation of the protein inph 4 solutions over the whole concentration range (100-250 mg/mL), whilethere almost no fragmentation detected at pH ranging from 5 to 8 overthe same concentration range. Consequently, the monomer content of pH 4solutions decreased accordingly (FIG. 30). Aggregate values were foundto increase with increasing pH values (from pH 4 to pH 8), independentof the concentration (FIG. 31).

17.10 Conclusions

This experiment was designed to examine the impact of solution pH andprotein concentration on viscosity and Dh (hydrodynamic diameter) ofAdalimumab solutions formulated in water by DF/UF processing. Suchsolutions are referred to as low-ionic solutions. A pH range of 4-8 wasevaluated, and protein concentrations tested were in a range between 2and 250 mg/mL.

With regard to viscosity (Section 17.4), it was found that low-ionicAdalimumab solutions have the same characteristics as Adalimumabsolutions formulated in the presence of ions (i.e. ionic excipients suchas organic buffer components or salts):

-   -   The higher the protein concentration, the higher solution        viscosity. This concentration-viscosity correlation was more        pronounced for solutions with pH values close to the Adalimumab        pI (i.e., pH 7 and pH 8). Conversely, for solutions at a        constant concentration, viscosity correlated with the closeness        of the solution's pH value to the pI of Adalimumab.

With regard to DLS data (Section 17.6), the following conclusions can bedrawn:

-   -   Adalimumab Dh values determined by DLS of low ionic Adalimumab        solutions were found to be lower than Dh values measured in        Adalimumab commercial formulations, especially at very low        solution pH.    -   The lower the solution pH, the lower Dh values determined by        DLS.    -   The higher the protein concentration, the lower the Dh values in        low-ionic Adalimumab solutions of a given pH.

The explanation for this behavior is that the ionic strength (i.e. thepresence of ions and ionizable excipients) in protein solutions iscrucial for the extent of protein-protein interactions. Especially atlower solution pH, charge-charge repulsions are more pronounced in lowionic Adalimumab solutions. When a protein is formulated in water byusing water as exchange medium in DF/UF processing, the amount ofionizable counter ions present that can compose both the Helmholtz layerand the Gouy-Chapman layer is notably reduced. Consequently,intermolecular charge-charge interactions (due to the charges of aminoacid residues present at the protein's surface) may be more pronouncedthan in an environment where ionizable counter ions (e.g. ionizableexcipients) are abundant, and charge-charge repulsion between proteinmonomers (leading to molecule motion in case of charge-charge repulsion)and random Brownian motion contribute to the mobility/motion of theprotein molecule measured by DLS. In DLS experiments, greater moleculemobilities are translated into greater molecular diffusion coefficients,which usually are assigned to molecules with smaller hydrodynamic sizesvia using the Stokes-Einstein equation. This can explain why thehydrodynamic diameter of proteins is reduced in low-ionic formulations.

Charge-charge interactions between antibody molecules can be repulsive(at lower solution pH) and attractive (at higher solution pH close tothe protein's pI).

17.11 Data Tables

TABLE 30 Adalimumab 100 mg/mL in buffer before DF versus water pH3 pH4pH5 pH6 pH7 pH8 pH9 turbity (NTU) 9.9 15.4 28.5 36.3 45.0 48.4 46.5viscosity (mPa * s) 2.5197 2.7935 3.2062 3.1512 3.5116 3.5494 3.5844viscosity (mm2/s) 2.4366 2.6991 3.0969 3.0444 3.3893 3.4261 3.4589density (g/cm3) 1.0341 1.0350 1.0353 1.0351 1.0361 1.0360 1.0363osmolality (mOsmol/kg) 293 287 298 297 286 279 285 Z-Ave d (nm) PCS 4.34.3 6.0 7.3 7.7 8.0 7.8 pH 4.00 4.99 6.00 7.03 8.00 9.03

TABLE 31 Adalimumab after DF versus water, before concentration, dilutedwith water to pH4 pH4 pH4 2 mg/mL 50 mg/mL 100.5 mg/mL turbity (NTU)0.296 1.46 3.30 viscosity (mPa*s) 0.9653 1.4471 2.2411 viscosity (mm2/s)0.9665 1.4298 2.1834 density (g/cm3) osmolality (mOsmol/kg) 40 Z-Ave d(nm) PCS 3.37 2.24 1.81 pH 4.29 Adalimumab after concentration anddilution with water to pH4 pH4 pH4 150.5 mg/mL 219.0 mg/mL 251.8 mg/mLturbity (NTU) 3.56 viscosity (mPa*s) 4.0283 13,,304 48.642 viscosity(mm2/s) 3.8712 12.614 45.567 density (g/cm3) osmolality (mOsmol/kg) 6496 141 Z-Ave d (nm) PCS 1.32 0.458 0.162 pH 4.32 4.54 Adalimumab afterDF versus water, before concentration, diluted with water to pH5 pH5 pH52 mg/mL 50 mg/mL 97.5 mg/mL turbity (NTU) 0.02 1.66 3.54 viscosity(mPa*s) 1.0563 1.6664 2.8661 viscosity (mm2/s) 1.0576 1.6465 2.7924density (g/cm3) 0.9988 1.0121 1.0264 osmolality (mOsmol/kg) 13 Z-Ave d(nm) PCS 157 32.4 1.3 pH 4.55 4.83 4.98 Adalimumab after concentrationand dilution with water to pH5 pH5 pH5 150.7 mg/mL 200.2 mg/mL 253.0mg/mL turbity (NTU) 7.24 viscosity (mPa*s) 7.0866 19.539 79.272viscosity (mm2/s) 6.8102 18.525 74.26 density (g/cm3) 1.0406 1.05471.0675 osmolality (mOsmol/kg) 78 80 96 Z-Ave d (nm) PCS 0.727 0.3350.255 pH 5.03 5.05 5.08

TABLE 32 Adalimumab after DF versus water, before concentration, dilutedwith water to pH6 pH6 pH6 2 mg/mL 50 mg/mL 100 mg/mL turbity (NTU) 0.4582.24 2.95 viscosity (mPa*s) 1.0696 1.8003 3.1147 viscosity (mm2/s)1.0708 1.7789 3.0385 density (g/cm3) 0.9989 1.012 1.0251 osmolality(mOsmol/kg) 3 + 11 = 14:2 = 7 27 11 Z-Ave d (nm) PCS 30.8 2.78 2.48 pH5.72 5.95 5.98

TABLE 33 Adalimumab after concentration and diluted with water to pH6pH6 pH6 146.6 mg/mL 201.8 mg/mL 248.5 mg/mL turbity (NTU) 9.29 viscosity(mPa*s) 9.0193 32.352 126.06 viscosity (mm2/s) 8.6775 30.709 118.07density (g/cm3) 1.0394 1.0535 1.0677 osmolality (mOsmol/kg) 37 58 95Z-Ave d (nm) PCS 0.989 0.355 0.108 pH 5.92 6.05 6.03

TABLE 34 Adalimumab after DF versus water, before concentration, dilutedwith water to pH7 pH7 pH7 2 mg/mL 50 mg/mL 103.2 mg/mL turbity (NTU) 0.17.13 14.9 viscosity (mPa*s) 1.1252 1.6898 4.2257 viscosity (mm2/s)1.1268 1.6688 4.1146 density (g/cm3) 0.9986 1.0126 1.027 osmolality(mOsmol/kg) 0 2 5 Z-Ave d (nm) PCS 3.31 4.16 2.89 pH 6.63 6.93 7.02

TABLE 35 Adalimumab after concentration and diluted with water to pH 7pH 7 pH 7 143.0 mg/mL 203.4 mg/mL 251.7 mg/mL turbity (NTU) 19.3viscosity (mPa*s) 14.024 74.987 343.881 viscosity (mm2/s) 13.492 70.928321.144 density (g/cm3) 1.0571 1.0708 osmolality (mOsmol/kg) 65 106 160Z-Ave d (nm) PCS 1.27 0.346 0.0876 pH 6.9 7.01 7.2

TABLE 36 Adalimumab after DF versus water, before concentration, dilutedwith water to pH 8 pH 8 pH 8 2 mg/mL 50 mg/mL 96.1 mg/mL turbity (NTU)0.41 12.10 28.300 viscosity (mPa*s) 1.261 1.8444 4.3486 viscosity(mm2/s) 1.2625 1.8224 4.2368 density (g/cm3) osmolality (mOsmol/kg) 5Z-Ave d (nm) PCS 5.59 5.62 4.28 pH 7.67

TABLE 37 Adalimumab after concentration and dilution with water to pH 8pH 8 pH 8 148.5 mg/mL 200.6 mg/mL 230.7 mg/mL turbity (NTU) 32.5viscosity (mPa*s) 20.102 85.5 233.14 viscosity (mm2/s) 19.318 81.066218.04 density (g/cm3) osmolality (mOsmol/kg) Z-Ave d (nm) PCS 1.420.398 0.168 pH 7.6

TABLE 38 PCS data: Adalmumab in buffer Pk1 Pk1 Pk2 Pk2 Pk3 Pk3 Z-Aved.nm PDI d.nm Area % d.nm Area % d.nm Area % pH 3 100 mg/mL 4.23 0.2834.43 86.4 54.1 13.6 0 0 pH 4 100 mg/mL 4.3 0.101 4.81 100 0 0 0 0 pH 5100 mg/mL 6.01 0.065 6.5 100 0 0 0 0 pH 6 100 mg/mL 7.25 0.063 7.82 1000 0 0 0 pH 7 100 mg/mL 7.64 0.094 8.53 100 0 0 0 0 pH 8 100 mg/mL 7.950.099 8.88 100 0 0 0 0 pH 9 100 mg/mL 7.7 0.133 8.98 100 0 0 0 0

TABLE 39 PCS data: Adalmumab in water Z-Ave d.nm PDI Pk1 d.nm Pk1 Area %Pk2 d.nm Pk2 Area % Pk3 d.nm Pk3 Area % pH 4 2 mg/ml 3.37 0.219 3.3988.8 73.3 11.2 0 0 pH 4 50 mg/ml 2.24 0.194 2.65 97.7 3300 2.3 0 0 pH 4100.5 mg/ml 1.81 0.172 2.02 97.4 3390 2.6 0 0 pH 4 150.5 mg/ml 1.320.181 1.64 100 0 0 0 0 pH 4 219.0 mg/ml 0.458 0.217 4070 62 0.621 38 0 0pH 4 251.8 mg/ml 0.162 0.263 0 0 0 0 0 0 pH 5 2 mg/ml 157 0.468 1.8884.3 181 10.7 17 5 pH 5 50 mg/ml 32.4 0.17 1.6 87.7 15.5 4.8 186 4.7 pH5 97.4 mg/ml 1.32 0.183 1.52 97.4 3290 2.6 0 0 pH 5 150.7 mg/ml 0.9310.209 1.36 98.7 3710 1.3 0 0 pH 5 200.2 mg/ml 0.335 0.203 0 0 0 0 0 0 pH5 253.0 mg/ml 0.107 0.255 0 0 0 0 0 0 pH 6 2 mg/ml 30.8 0.382 2.78 60.9273 30.2 5070 5 pH 6 50 mg/ml 2.78 0.247 2.68 86.4 1600 7.8 114 5.8 pH 6100 mg/ml 2.01 0.171 2.48 100 0 0 0 0 pH 6 146.6 mg/ml 0.989 0.219 1.3296.9 3770 301 0 0 pH 6 201.8 mg/ml 0.355 0.231 0 0 0 0 0 0 pH 6 248.5mg/ml 0.108 0.301 0 0 0 0 0 0 pH 7 2 mg/ml 3.31 0.211 3.58 93.9 1250 6.10 0 pH 7 50 mg/ml 4.16 0.132 4.84 100 0 0 0 0 pH 7 103.2 mg/ml 2.890.141 3.39 100 0 0 0 0 pH 7 143.3 mg/ml 1.27 0.212 1.68 100 0 0 0 0 pH 7203.4 mg/ml 0.346 0.306 0 0 0 0 0 0 pH 7 251.7 mg/ml 0.0876 0.497 0 0 00 0 0 pH 8 2 mg/ml 5.59 0.365 3.15 67.4 244 30.2 26.5 2.4 pH 8 50 mg/ml5.62 0.174 7 100 0 0 0 0 pH 8 96.1 mg/ml 4.28 0.192 4.81 96.9 3640 3.1 00 pH 8 148.5 mg/ml 1.43 0.253 1.68 93.9 2910 6.1 0 0 pH 8 200.6 mg/ml0.398 0.246 4920 100 0 0 0 0 pH 8 230.7 mg/ml 0.168 0.3 0 0 0 0 0 0

TABLE 40 SEC data pH conc. mg/mL % aggregate % monomer % fragmente Area(mVs) 4 100 0.28 67.95 31.76 45195.348 4 150 0.26 66.07 33.68 44492.8034 200 0.30 64.59 35.11 52558.050 4 250 0.29 64.40 35.31 48491.299 5 1001.46 98.44 0.11 48127.249 5 150 1.33 98.56 0.11 43226.397 5 200 1.3998.50 0.11 43634.282 5 250 1.38 98.52 0.11 41643.062 6 100 2.00 97.900.10 44338.373 6 150 2.52 97.37 0.11 41899.182 6 200 2.52 97.37 0.1143869.183 6 250 2.39 97.50 0.11 34969.456 7 100 2.78 97.12 0.1046194.824 7 150 4.24 95.65 0.11 47443.014 7 200 3.61 96.29 0.1041916.220 7 250 3.39 96.50 0.11 38185.208 8 100 3.24 96.65 0.1242334.491 8 150 3.64 96.18 0.18 40305.890 8 200 3.63 96.25 0.1340280.342 8 250 3.76 96.05 0.19 32067.297

Example 18: Impact of pH on J695 Viscosity

Viscosity data were generated for J695 after DF/UF processing usingwater as exchange medium. J695 DS (see Example 1) was diafilteredagainst water, applying at least 5 DF/UF steps. Viscosity was thendetermined at various temperatures using a plate-plate viscometer, 100rpm shear rate, 150 μm gap, 60 mm plate diameter (equipment: BohlinGeminim viscometer (Malvern Instruments, Southborough, Mass.),temperature range evaluated 8-25° C.).

As seen in FIG. 32, at concentrations of 179 mg/mL and 192 mg/mL,respectively, J695 solution viscosities were below 70 cP at 12° C.,below 40 cP at 20° C., and below 30 cP at 25° C.

Example 19: Pharmacokinetics (PK) of an Antibody in Pure Water

The goal of this study was to evaluate potential impact of formulationparameters (i.e. low ionic protein formulation containing water vsconventional protein formulations using ionic excipients such as buffersand salts) on local tolerability and PK after sub-cutaneous (s.c.)dosing with Afelimomab. In addition, systemic toxicity and toxicokineticdata of the formulations was investigated. Protein concentrations usedranged from 50 mg/mL to 200 mg/mL and ionic strengths ranged from 3mOsm/kg to 300 mOsm/kg.

A single (s.c.) dose feasibility study was carried out with Afelimomab(MAK195F—mouse anti human TNF F(ab′)2 (Abbott Laboratories)) in maleSprague-Dawley rats to assess the local tolerance and toxicity ofAfelimomab in rats following s.c. administration of liquid formulationsat 50 and 200 mg/kg. Single s.c. doses were followed by anobservation/recovery period. Limited blood sampling was carried out tomeasure circulating Afelimomab levels and assess absorption andhalf-life. The administered dose volume was 1 mL/kg body weight. Theexperimental groups included the following:

Experimental Groups 01 Control (vehicle) 02 50 mg/ml Afelimomab, liquid,standard formulation 07 200 mg/ml Afelimomab, liquid, water formulationGroup A Observation period 2 days Group B Observation period 7 daysGroup C Observation period 14 daysGrouping and Rat Identification (N=1 Per Group)

Animal number Group Group A Group B Group C 01 1 2 3 02 4 5 6 07 19 2021

The animals were repeatedly observed for clinical signs and mortality onday 1 at 15 min, 1, 3, 5, and 24 hours past administration and at leastonce daily afterwards. Body weights were measured on the days of dosing(day 1) and necropsy (day 3, 15 or 21, respectively) and twice weekly,if applicable. Blood samples for drug analysis were collected on Day 1(4 hours past administration), and on Days 2, 3, 5, 8, and 15 asapplicable. Prior to necropsy blood was collected and hematological andclinical chemistry parameters were evaluated. Blood smears were preparedof each animal prior to necropsy. At necropsy, macroscopy of bodycavities was performed. Organ weight measurement was performed on liver,kidneys, thymus, spleen, and lymph nodes. Preliminary histopathology wasperformed on the injection site and on liver, kidneys, thymus, spleen,and lymph nodes.

All animals survived the study until scheduled necropsy. The ratadministered the water formulation showed crusts in the cervical regionfrom Day 14 to 15. No test item-related effect on body weight wasobserved. Hematology and clinical chemistry values were variable. Noclearly test item-related changes were identified in haematology orclinical chemistry. No test item-related changes were noted inurinalysis. Measurement of organ weights resulted in high variabilityand no clearly test item-related changes in organ weights.

At gross observation reddening of the subcutis at the area of injectionwas noted in the rat receiving the water formulation at Day 3. All otherchanges belonged to the spectrum of spontaneous findings commonly seenin Sprague-Dawley rats of this strain and age.

Microscopic Findings were as follows:

-   -   No findings in Groups 01, 02    -   Minimal diffuse subcutaneous inflammation in Group 07    -   Focal subcutaneous hemorrhage correlating with reddening on        gross pathology in Group 07 (Day 3), thought to be        administration related    -   Preliminary immunohistochemistry results of pan-T,        suppressor/cytotoxic T cells/natural killer cells, pan-B cells        and pan-macrophage markers on the local reactions indicate        mainly macrophages and natural killer cells involved in the        subcutaneous inflammations/infiltrations. Thus, so far there are        no hints for a local immunogenic response to the formulations        used.

All other changes belonged to the spectrum of spontaneous findingscommonly seen in Sprague-Dawley rats of this strain and age.

Following subcutaneous administration Afelimomab absorption appeared tobe fast with maximum serum levels reached 0.2-3 days after injection.The absolute levels of Afelimomab in all samples tested were low. Largevariability was observed between the samples, likely because of thelimited sampling frequency and the low number of animals used. In mostsamples, no Afelimomab could be detected in serum after 5-8 days. Thisdrop in serum levels is probably due to the high clearance of theF(ab′)2. The observed T½ for most samples were in the range of 1-2 daysin agreement with previous observations. The longer half-life of thelow-ionic formulation (7.8 d) may represent a protracted absorption ofthe sample. Data are presented in Tables 41 and 42.

TABLE 41 Plasma exposure levels of MAK195F 50 mg/kg liquid standardformulation Time Concentration (μg/ml) Average (day) Rat 4 Rat 5 Rat 6(μg/ml) STD 0.167 1.40 1.17 1.38 1.32 0.13 2 0.76 0.97 0.66 0.80 0.16 30.45 0.67 0.47 0.53 0.12 5 LLOQ LLOQ LLOQ 8 LLOQ LLOQ LLOQ 15 LLOQ LLOQ200 mg/kg water formulation Time Concentration (μg/ml) Average (day) Rat19 Rat 20 Rat 21 (μg/ml) STD 0.167 1.27 3.01 3.17 2.48 1.05 2 0.17 1.571.53 1.09 0.80 3 LLOQ 1.54 1.56 1.55 0.02 5 0.64 0.66 0.65 0.02 8 0.400.37 0.38 0.02 15 0.25 0.25 0.00 LLOQ = below quantitation limit

There were no aggregation state findings for liquids, neither Afelimomabor control substance.

For the low-ionic strength formulation, minimal diffuse s.c. injectionsite inflammation was seen. Inflammation, either minimal to slight, orslight to moderate, was seen with increased protein concentration (50mg/mL and 200 mg/mL, respectively). Some local s.c. hemorrhage was seen,correlating with reddening on gross pathology; this was considered to bethe consequence of blood vessel puncture during injection. Somesubcutaneous reddening at the injection site was observed at Day 3 forthe water formulation, but was not considered detrimental. Overall, theformulation was tolerated locally.

In Table 42 below, PK data of conventional liquid formulation vs. thewater formulation is presented.

TABLE 42 Pharmacokinetic parameters of MAK 195F after subcutaneousdosing in different formulations. Last AUC Mean Time of Last DetectableDose Half-life Tmax Cmax (day * Residence Detectable Conc. (mg/kg)Formulation (day) (day) (μg/ml) μg/ml) Time (day) Conc. (day) (μg/ml) 50liquid 0.5 0.2 1.32 3.3 1.5 3 0.53 200 water 5.9 0.2 2.48 11.8 7.5 150.25 formulationAn increase in duration of detectable serum levels was seen with lowionic formulation (i.e. Afelimomab formulated in water), as seen inTable 42.

In this study, the observed absolute levels of MAK195F in low ionicsolution (water) provided a better exposure, longer detectable serumlevels and ‘half-life’ than in conventional MAK195F liquid formulation.

Afelimomab half-lives were in the range of 1-2 days in standardformulation in agreement with previous observations for F(ab′)₂molecules. However, a seemingly longer half-life was observed for thelow-ionic formulation (7.8 d). Accordingly, the mean residence time ofMAK 195F in this formulation appeared to be longer compared to thestandard formulation tested.

Example 20: DF/UF of 2.5(E)mg1 (Anti IL-18 Antibody)

Diafiltration/ultrafiltration (continuous mode) of 2.5(E)mg1 bulksolution (59.6 mg/mL) was performed, applying an about 4-fold volumeexchange using water for injection (in the following referred to as“water”). The DF/UF operation was controlled by monitoring turbidity,protein concentration (OD280), pH and osmolarity of retentate, and DLSmeasurements. During DF/UF, permeate osmolarities were also monitored tocontrol the excipient reduction of the 2.5(E)mg1 bulk solutions.

Materials and Methods

2.5(E)mg1 Bulk Drug Substance (methionine, histidine, free ofpolysorbate 80) (Abbott Bioresearch Center, Worcester, Mass.): 2 PETGbottles with a total of 589.12 g solution, solution concentration 59.6mg/mL.

-   -   Ampuwa (water for injection) (Fresenius Medical Care, Waltham,        Mass.).    -   Millipore Labscale TFF DF/UF unit including 2× Pellicon XL        filter cassettes, Millipore, PLCTK 30 kDa membrane, regenerated        cellulose    -   UV/VIS spectrophotometer, Specord 50 using 280 nm wavelength    -   Metrohm pH-meter, type 744 with Biotrode probe No. 57    -   Osmometer: Knauer, K-7400    -   density measurements using equipment of Paar, DMA 4100    -   Laminar air flow box Hereaus    -   turbidity measurements: Hach, 2100AN    -   viscometer: Paar, AMVn    -   scales: Mettler Toledo, AT261 and 33.45    -   filters: Millex AP 20 (fiberglass) and Minisart High Flow Filter        (cellulose acetate), 0.20 μm pore size.        20.1 Experimental Procedures

Thawing of 2.5(E)mg1 DS samples: 2 L PETG bottles containing frozen DSwere thawed within 2 hrs using a circulating water bath at 23° C. Thethawed DS was clear, slightly opalescent, and free from visibleparticles.

Concentration of DS by DF/UF: due to the DF/UF unit reservoir volumelimit of 530 mL, the 2.5(E)mg1 DS was concentrated to a final volume of525 mL.

DF/UF using water (buffer exchange): the DS (methionine, histidine,2.5(E)mg1) was subjected to DF/UF, applying a 4-fold volume exchange.Table 43 gives the amounts of water that were used throughout theexperiment and Table 44 provides the experimental parameters.

TABLE 43 DF/UF water volume exchanges Volume of water used Volumeexchange (cumulative) 1-fold  576 mL 2-fold 1152 mL 3-fold 1728 mL4-fold (end of experiment) 2304 mL

TABLE 44 DF/UF procedure parameters Labscale TFF DF settings Pump speed1.5-2   Pressure of pump 20-30 psi Stirring speed ~3 Experiment duration8 hrs

Osmolarity measurement of permeate was performed at about every 200 mLof permeate processed.

After DF/UF against water, the volume of the 2.5(E)mg1 solution was 450mL and the protein concentration 76.6 mg/mL. This solution, containing2.5(E)mg1 dissolved essentially in water, was then concentrated.

TABLE 45 DF/UF process parameters for solution concentration LabscaleTFF UF settings Pump speed 1.5-2 Pressure of pump max. 30 psi Stirringspeed ~3 Experiment duration 51 min. Final weight of 257.83 g solution:

The concentrated solution (˜130 mg/mL) was subjected to 0.2 μmfiltration. The solution was cooled to 2-8° C. and then stored at −80°C.

20.2 Data Collected During DF/UF of 2.5(E)Mg1

TABLE 46 In Process control data Temperature solution/ Osmo- Room lalityconc DF Volume temperature turbidity [mOsmol/ [mg/ steps [mL] time [°C.] [NTU] pH kg] ml] 2.5(E)mg1 14.5 5.91 150 59.6 0¹ 0 08:00 19.0/24.1N/A 5.91 125 65.2 1 575 10:02 24.4/24.4 10.1 5.92 50 70.1 2 1150 11:5024.3/24.7 6.67 5.94 16 72.8 3 1730 13:50 25.0/24.8 6.55 5.97 6 74.6 ca.4 2200 15:35 25.8/25.5 10.1 5.97 5 76.7

TABLE 47 Osmolalty of permeate (fractionated and measured duringprocess) Tem- perature solution/ No. Room Of osmo- tem- Permeat DF/lality Sample perature Permeate cumulative UF [mOsmol/ no. time [° C.][ml] [ml] steps kg]  0³ 07:35 N/A 90 N/A N/A 125 1 08:00 19.0/24.1 200200 0.3 124 2 08:50 23.0/24.1 200 400 0.7 82 3 09:27 24.0/24.2 200 6001.0 53 4 10:12 24.4/24.4 200 800 1.4 37 5 10:50 24.5/24.3 200 1000 1.725 6 11:25 24.6/24.3 200 1200 2.1 16 7 12:10 24.7/24.3 230 1430 2.5 7 812:55 24.7/24.4 170 1600 2.8 4 9 13:25 24.8/24.4 200 1800 3.1 2 10 14:15 25.1/24.8 200 2000 3.5 0 11  14:55 25.8/25.5 200 2200 3.8 1

TABLE 48 Concentration of 2.5(E)mg1 solution after buffer exchangeSolution Volume in reservoir (i.e. temperature retentate) time [° C.][ml] pH 15:54 25.9 450 5.94 16:02 26.1 400 5.96 16:07 26.1 375 5.9616:20 26.2 350 5.96 16:28 26.3 300 5.98 16:35 26.5 275 5.98 16:45 26.5250 5.99

TABLE 49 Analytical characterization of concentrated 2.5(E)mg1 solution(before and after 0.2 μm filtration): lot parameter before filtrationafter filtration turbidity 15.4 9.58 [NTU] osmolality 6 N/A [mOsmol/kg]density 1.0346 N/A [g/ml] pH 5.99 N/A Dyn. Viscosity N/A 7.9998 (° C.)[mPas]

TABLE 50 Dynamic light scattering data (determination of Dh of monomerand z-average value of Dh = Dh of all specimen present in solution)during DF/UF Sample pull DV DV DV DV After After 1- 2- 3- 4- con-filtra- DLS data fold fold fold fold centration tion Peak 1 diameter4.32 3.68 3.54 3.48 2.03 2.13 monomer 100.0 100.0 100.0 89.6 87.0 100.0[nm] intensity 3.95 3.28 3.20 3.53 2.12 1.89 [%] Z-Average 0.077 0.1060.094 0.245 0.287 0.113 [nm] Pdl Peak 2 diameter N/A N/A N/A 984 411 N/A[nm] intensity 10.4 11.8 [%] Peak 3 diameter N/A N/A N/A N/A 4260 N/A[nm] intensity 1.2 [%]20.3 Discussion

The experiment demonstrated that 2.5(E)mg1 (buffered in methionine,histidine) can be formulated in essentially water at higherconcentration (no solubility limitations observed at 130 mg/mL). After 3volume exchanges using water osmolality of permeate and retentate werebelow 10 mOsmol/kg, demonstrating that buffer excipients have beeneffectively reduced. The opalescence of the 2.5(E)mg1 solution wasreduced during DF/UF using water (optimal appearance), mirrored also byreduces turbidity values (nephelometric turbidity units (NTU) of DSstarting solution 14.5, after 3-fold volume exchange 6.55, after 4-foldvolume exchange 10.5.

As seen with other antibodies, the hydrodynamic diameter as determinedby DLS decreased due to excipient reduction (intermolecularcharge-charge repulsion adding to random Brownian motion, resulting inhigher molecule mobility, translates to lower Dh values calculated). ThepH of the 2.5(E)mg1 solution was basically the same before (pH 5.94) andafter (pH 5.99) the DF/UF operation.

As shown by DLS monitoring, the 2.5(E)mg1 remained stable during theDF/UF operation. No substantial increase in high molecular weightspecimen was detected.

Example 21: Preparation of Adalimumab Formulated in Water and StabilityStudies Thereof

The following example describes the stability of a formulationcomprising adalimumab originating from processes described in the aboveexamples, i.e., adalimumab was successfully dialyzed into water.

Materials and Methods

3323.6 g Adalimumab solution (71.3 mg/mL) were diafiltered using purewater. After a 7-fold volume exchange with pure water (theoreticalexcipients reduction, 99.9%), the protein solution wasdiluted/ultrafiltered to final target concentrations of 220 and 63mg/mL, respectively. PH, osmolality, viscosity, conductivity, PCS,visual inspection and protein concentration measurements (OD280) wereperformed to monitor the status of the protein after DF/UF processing.

After DF/UF processing, the protein solutions were sterile filtered(0.22 μm Millipak-60 and Millipak-200 membrane filters) and subsequentlyfilled into BD HyPak SCF™ 1 mL long syringes, equipped with 27.5 G RNSneedles and sterile BD HyPak BSCF 4432/50 stoppers. The filling volumewas around 0.825 mL per syringe.

After filling the syringes were stored at 2-8° C., 25° C. and 40° C.,respectively, and analyzed as indicated in the sample pull schemedepicted below.

-   -   Adalimumab Drug Substance (Adalimumab extinction coefficient 280        nm: 1.39 mL/mg cm): Drug Substance did not contain        polysorbate 80. DS buffer, pH 5.38.    -   Sortorius Sartocon Slice diafiltration system, equipped with        Ultrasert PES membrane cassettes (50 kDa and 30 kDa cutoff). The        Sartocon Slice system was operated in continuous mode at ambient        temperature according to Sartorius Operating Instructions.    -   pH electrodes    -   PerkinElmer UV visible spectrophotometer, Lambda 35, was used        for protein concentration measurements (280 nm wavelength).        Disposable UV cuvettes, 1.5 mL, semi-micro, Poly(methyl        methacrylate) (PMMA), were used for the concentration        measurements.    -   Sterilized water for injection Ph.Eur./USP was used as DF/UF        medium.    -   A Vogel Osmometer OM815, was used for osmolality measurements        (calibrated with 400 mOsmol/kg NaCl calibration solution, Art.        No. Y1241, Herbert Knauer GmbH, Berlin, Germany).    -   Anton Paar Microviscosimeter, type AWVn, was used for viscosity        assessment of the protein solutions according to Anton Paar        Operating Instructions. Viscosity was assessed at 20° C.    -   An InoLab Cond Level2 WTW device was used for conductivity        measurements normalized to 25° C.    -   A Malvern Instruments Zetasizer nano ZS, was used for        determination of Z-average values, applying a standard method.        Measurements were performed at 25° C., using viscosity data        obtained by falling ball viscosimetry (Anton Paar        Microviscosimeter, type AWVn, at 25° C.).

HPLC Methods

-   -   Adalimumab, SEC analysis: Sephadex 200 column (Pharmacia Cat.        No. 175175-01). Mobile phase 20 mM sodium phosphate, 150 mM        sodium chloride, pH 7.5, 0.5 mL/min flow rate, ambient        temperature, detection UV 214 nm and 280 nm. Each sample was        diluted to 1.0 mg/mL with Milli-Q water, sample injection load        50 (duplicate injection).    -   Adalimumab, IEC analysis: Dionex, Propac WCX-10 column        (p/n 054993) along with a corresponding guard column (p/n        054994). Separation conditions: mobile phase A: 10 mM sodium        phosphate, pH 7.5; mobile phase B 10 mM Sodium phosphate, 500 mM        Sodium chloride, pH 5.5. 1.0 mL/min flow rate, ambient        temperature. Each sample was diluted to 1.0 mg/mL with Milli-Q        water, sample injection load 100 μg, duplicate injection.        Calculation of the Protein Concentration

Calculation Formula:

$E = {{{- l}{g\left( \frac{I}{I_{0}} \right)}} = {\left. {ɛ \cdot c \cdot d}\rightarrow c \right. = \frac{E}{ɛ \times d}}}$ɛ − absorption  coefficientc − concentrationd − length  of  cuvette  that  the  light  has  to  passE − absorbanceI₀ − initial  light  intensityI − light  intensity  after  passing  through  sample$ɛ_{Adalimumab} = {1.39\frac{mL}{{mg} \times {cm}}}$Sample Pull SchemeSamples of the prepared solutions are stored at the temperatures listedbelow and pulled (x) at the indicated time points after study start.

Temp. T0 1 m 3 m  5° C. — x x 25° C. x x x 40° C. — x x

Test parameter Test method Visible particles analogous DAC (EA 4.43)Subvisible particles analogous Ph.Eur./USP EA 4.44 Turbidity analogousPh.Eur. (EA 4.42) Color (visual) Ph.Eur. (EA 4.50) pH Ph.Eur. (EA 4.24)Size exclusion HPLC Desribed in the text above Cation exchange HPLCDesribed in the text aboveDF/UF Processing of Adalimumab

Table 51 describes the adalimumab characteristics after diafiltration.

TABLE 51 Protein PCS Concentration Osmolality Viscosity VisualConductivity [Z-average/ Sample [mg/mL] pH [mosmol/kg] [cP] Inspection[μS/cm] d · nm] High 220 5.57 26 27.9 Slightly 1167 0.34 concentrationopalescent, essentially free from visible particles Low 63 5.44 5 1.8Slightly 522 1.85 concentration opalescent, essentially free fromvisible particlesAdalimumab characterization upon storage, including clarity andopalescence, degree of coloration of liquids, SEC, at differenttemperature degrees is described in Appendix D.Conclusion

The above example provides a diafiltration/ultrafiltration (DF/UF)experiment where water (sterilized water for injection Ph.Eur./USP) wasused as diafiltration medium for the monoclonal antibody Adalimumab.

Adalimumab was subjected to DF/UF processing by using pure water asDF/UF exchange medium and was formulated at pH 5.57 at highconcentration (220 mg/mL) and at pH 5.44 at lower concentration (63mg/mL) without inducing solution haziness, severe opalescence orturbidity formation.

Adalimumab from the DF/UF experiments was stored in SCF syringes at 2-8°C., 25° C. and 40° C. for 3 months. Data obtained points at favorableoverall stability of the protein.

In conclusion, processing and formulating proteins using pure water asDF/UF exchange medium is feasible. Assuming an ideal 100% excipientmembrane permeability, an approx. 99.9% maximum excipient reduction canbe estimated.

Example 22: Stability Studies of Adalimumab Formulated in Water UsingNon-Ionic Excipients

The following example describes stability studies of a formulationcontaining an antibody, i.e., adalimumab, in water with additionalnon-ionic excipients.

Materials and Methods

Adalimumab material was the same as in example 21 (DF/UF processing).After DF/UF processing, the protein solutions were formulated as denotedin Table 52. Mannitol was chosen as example from the group of sugaralcohols, like mannitol, sorbitol, etc. Sucrose was chosen as examplefrom the group of sugars, like sucrose, trehalose, raffinose, maltose,etc. Polysorbate 80 was chosen as example from the group of non-ionicsurfactants, like polysorbate 80, polysorbate 20, pluronic F68, etc.˜10.7 mL were prepared for any formulation. Osmolality, viscosity andPCS measurements were performed for any formulation after preparation.

TABLE 52 Final protein Mannitol Sucrose Polysorbate Sample concentration(mg/mL) (mg/mL) 80 (% w/w) Name 50 mg/mL 50 — — LI 50/01 50 mg/mL — 80 —LI 50/02 50 mg/mL 50 — 0.01 LI 50/03 50 mg/mL — 80 0.01 LI 50/04 50mg/mL 50 — 0.1 LI 50/05 50 mg/mL — 80 0.1 LI 50/06 50 mg/mL — — 0.01 LI50/07 50 mg/mL — — 0.1 LI 50/08 200 mg/mL 50 — — LI 200/01 200 mg/mL —80 — LI 200/02 200 mg/mL 50 — 0.01 LI 200/03 200 mg/mL — 80 0.01 LI200/04 200 mg/mL 50 — 0.1 LI 200/05 200 mg/mL — 80 0.1 LI 200/06 200mg/mL — — 0.01 LI 200/07 200 mg/mL — — 0.1 LI 200/08

-   -   Polysorbate 80 stock solution 0.5% and 5% in sterile water for        injection: Addition in 1:50 ratio (210 μL to 10.5 mL Adalimumab        solution, addition of 210 μL water for injection to samples        formulated without surfactant to assure equal protein        concentration in all samples)    -   Addition of mannitol/sucrose in solid form (525 mg/840 mg,        respectively).

The preparations were sterile filtered (Millex GV, Millipore, 0.22 μm, Ø33 mm, Art. SLGV033RS) and subsequently filled into BD HyPak SCF™ 1 mLlong syringes, equipped with 27.5 G RNS needles and sterile BD HyPakBSCF 4432/50 stoppers. The filling volume was around 0.6 mL per syringe.

After filling the syringes were stored at 2-8° C., 25° C. and 40° C.,respectively, and analyzed as indicated in the sample pull schemedepicted below.

-   -   Adalimumab Drug Substance (Adalimumab extinction coefficient 280        nm: 1.39 mL/mg cm): Drug Substance did not contain        polysorbate 80. DS buffer, pH 5.38.    -   PH electrodes    -   Sterilized water for injection Ph.Eur./USP was used as DF/UF        medium.    -   Mannitol, polysorbate 80, and sucrose, all matching Ph.Eur.        quality    -   A Vogel Osmometer OM815, was used for osmolality measurements        (calibrated with 400 mOsmol/kg NaCl calibration solution, Art.        No. Y1241, Herbert Knauer GmbH, Berlin, Germany).    -   Anton Paar Microviscosimeter, type AWVn, was used for viscosity        assessment of the protein solutions according to Anton Paar        Operating Instructions. Viscosity was assessed at 20° C.    -   Fluostar Optima, BMG Labtech (absorption measurement at 344 nm        in well plates, assessment of turbidity)    -   A Malvern Instruments Zetasizer nano ZS, was used for        determination of Z-average values, applying a standard method.        Measurements were performed at 25° C., using viscosity data        obtained by falling ball viscosimetry (Anton Paar        Microviscosimeter, type AWVn, at 25° C.).        HPLC Methods    -   Adalimumab, SEC analysis: Sephadex 200 column (Pharmacia Cat.        No. 175175-01). Mobile phase 20 mM sodium phosphate, 150 mM        sodium chloride, pH 7.5, 0.5 mL/min flow rate, ambient        temperature, detection UV 214 nm and 280 nm. Each sample was        diluted to 1.0 mg/mL with Milli-Q water, sample injection load        50 μg (duplicate injection).    -   Adalimumab, IEC analysis: Dionex, Propac WCX-10 column along        with a corresponding guard column. Separation conditions: mobile        phase A: 10 mM sodium phosphate, pH 7.5; mobile phase B 10 mM        Sodium phosphate, 500 mM Sodium chloride, pH 5.5. 1.0 mL/min        flow rate, ambient temperature. Each sample was diluted to 1.0        mg/mL with Milli-Q water, sample injection load 100 μg,        duplicate injection.        Sample Pull Scheme

Samples of the prepared solutions were stored at 5° C., 25° C., and 40°C. and pulled at either 1 minute (5° C. and 40° C.) or at T0 and 1minute (25° C.) after study start. Test parameters were measuredaccording to appropriate methods, e.g., color was determined visually,turbidity was determined at an absorption at 344 nm.

Initial Formulation Characterization

Table 53 described the initial formulation osmolarities and viscosities.

TABLE 53 osmolarity viscosity Lot. comp. rmosmoll rmPasI LI 50/01 50mg/mL mannitol 309 1.9796 LI 50/02 80 mg/mL sucrose 272 2.1284 LI 50/0350 mg/mL mannitol; 0.01% Tween 80 307 1.9843 LI 50/04 80 mg/mL sucrose;0.01% Tween 80 269 2.1194 LI 50/05 50 mg/mL mannitol; 0.1% Tween 80 3071.9980 LI 50/06 80 mg/mL sucrose; 0.1% Tween 80 272 2.1235 LI 50/070.01% Tween 80 8 1.7335 LI 50/08 0.1% Tween 80 8 1.8162 LI 200/01 50mg/mL mannitol 396 21.395 LI 200/02 80 mg/mL sucrose 351 21.744 LI200/03 50 mg/mL mannitol; 0.01% Tween 80 387 21.233 LI 200/04 80 mg/mLsucrose; 0.01% Tween 80 350 21.701 LI 200/05 50 mg/mL mannitol; 0.1%Tween 80 387 21.592 LI 200/06 80 mg/mL sucrose; 0.1% Tween 80 355 21.943LI 200/07 0.01% Tween 80 27 21.296 LI 200/08 0.1% Tween 80 28 21.889

All formulations of one concentration demonstrated equal viscosities.Those of sucrose containing formulations were slightly higher. Thereduced viscosities of the highly concentrated formulations incomparison to the highly concentrated formulation in water (example A,viscosity 27.9 cP) is explained by sample dilution with polysorbate 80stock solutions or plain water, leading to a final concentration of ˜215mg/mL vs. 220 mg/mL in example 21.

Table 54 describes the PCS data determined for each sample.

TABLE 54 PCS Sample [Z-average/d · nm] LI50/01 2.58 LI50/02 2.22 LI50/032.13 LI50/04 2.22 LI50/05 2.25 LI50/06 2.55 LI50/07 2.87 LI50/08 1.94LI200/01 0.50 LI200/02 0.43 LI200/03 0.36 LI200/04 0.38 LI200/05 0.37LI200/06 0.41 LI200/07 0.35 LI200/08 0.36The data provided in Table 54 shows that z-average values do notsignificantly differ from the values obtained from Adalimumab solutionsin non-ionic excipient free systems (63 mg/mL, 1.85 d·nm, 220 mg/mL,0.34 d·nm, example 21).Adalimumab Characterization Upon Storage

Appendix E provides data on Adalimumab stability upon storage.

Conductivity of Placebo Solutions

Table 55 describes the influence of non-ionic excipients on theconductivity of the various adalimumab formulations. All placebosolutions were prepared using sterilized water for injectionPh.Eur./USP.

TABLE 55 Polysorbate 80 Conductivity Mannitol (mg/mL) Sucrose (mg/mL) (%w/w) (μS/cm) — — — 1.1 50 — — 1.2 — 80 — 2.2 50 — 0.01 2.3 — 80 0.01 1.450 — 0.1 2.6 — 80 0.1 3.6 — — 0.01 1.2 — — 0.1 2.6Conclusion

The preparations were stored in SCF syringes at 2-8° C., 25° C. and 40°C. for 1 month. Data obtained from the storage study showed that therewas overall stability of the protein in all formulations tested. Thestability data was comparable to the stability of samples from example21. Measurement of the conductivity of non-ionic excipient containingplacebo solutions demonstrates a marginal increase of conductivity forsome excipients in the range of some μS/cm. PCS measurements demonstrateno significant increase in hydrodynamic diameters in comparison tonon-ionic excipient free systems.

In conclusion, processing proteins using pure water as DF/UF exchangemedium and formulation with non-ionic excipients is feasible. Adalimumabwas also assessed by PCS in a buffer of following composition: 10 mMphosphate buffer, 100 mM sodium chloride, 10 mM citrate buffer, 12 mg/mLmannitol, 0.1% polysorbate 80, pH 5.2. The Adalimumab concentration was50 mg/mL and 200 mg/mL, respectively. The z-average values were 11.9d·nm for the 50 mg/mL formulation and 1.01 d·nm for the 200 mg/mLformulation, respectively. Thus, it was clearly demonstrated thathydrodynamic diameters at a given protein concentration are dependent onthe ionic strength (clearly higher diameters in salt containingbuffers).

Example 23: Preparation of J695 Formulated in Water with Non-IonicExcipients

The following example describes the preparation of a formulationcontaining an antibody, i.e., adalimumab, in water with additionalnon-ionic excipients. The example also describes the stability (asmeasured for example by SE-HPLC and IEC) of J695 formulated in waterwith additional non-ionic excipients.

Materials and Methods

2×30 mL J695 solution (˜125 mg/mL) at different pH were dialyzed usingpure water applying Slide-A-Lyzer dialysis cassettes. Dialysis of thesamples was performed for 3 times against 3 L pure water, respectively(theoretical excipients reduction, 1:1,000,000). The protein solutionswere ultrafiltered to final target concentrations of 200 mg/mL, by usingVivaspin 20 concentrators. PH, osmolality, viscosity, conductivity, PCS,visual inspection, HPLC and protein concentration measurements (OD280)were performed to monitor the status of the protein during and afterprocessing.

After processing, the protein solutions were formulated as denoted inthe following. Mannitol was chosen as an example to use from the groupof sugar alcohols, like mannitol, sorbitol, etc. Sucrose was chosen asan example to use from the group of sugars, like sucrose, trehalose,raffinose, maltose, etc. Polysorbate 80 was chosen as an example to usefrom the group of non-ionic surfactants, like polysorbate 80,polysorbate 20, pluronic F68, etc. A volume of 0.5 mL was prepared foreach of these formulations. PH, osmolality, visual inspection, and HPLCanalysis were performed to monitor the status of the protein aftersample preparation.

TABLE 56 Description of various J695 formulations Final protein MannitolSucrose Polysorbate 80 concentration (mg/mL) (mg/mL) (% w/w) SampleName* 200 mg/mL 50 — — LI200/01/5 200 mg/mL — 80 — LI200/02/5 200 mg/mL50 — 0.01 LI200/03/5 200 mg/mL — 80 0.01 LI200/04/5 200 mg/mL 50 — 0.1LI200/05/5 200 mg/mL — 80 0.1 LI200/06/5 200 mg/mL — — 0.01 LI200/07/5200 mg/mL — — 0.1 LI200/08/5 200 mg/mL 50 — — LI200/01/6 200 mg/mL — 80— LI200/02/6 200 mg/mL 50 — 0.01 LI200/03/6 200 mg/mL — 80 0.01LI200/04/6 200 mg/mL 50 — 0.1 LI200/05/6 200 mg/mL — 80 0.1 LI200/06/6200 mg/mL — — 0.01 LI200/07/6 200 mg/mL — — 0.1 LI200/08/6 *The term“/5” or “/6” is added to any sample name to differentiate betweensamples at pH 5 and 6.

-   -   Polysorbate 80 stock solution 0.5% and 5% in sterile water for        injection:    -   Addition in 1:50 ratio (10 μL to 0.5 mL J695 solution, addition        of 10 μL water for injection to samples formulated without        surfactant to assure equal protein concentration in all samples)    -   Addition of mannitol/sucrose in solid form (25 mg/40 mg,        respectively).    -   J695 Drug Substance (J695 extinction coefficient 280 nm: 1.42        mL/mg cm): Drug Substance did not contain polysorbate 80. DS        buffer, pH 6.29.    -   pH electrodes    -   Demineralized and sterile filtered water was used as dialysis        medium.    -   Mannitol, polysorbate 80, and sucrose, all matching Ph.Eur.        quality    -   A Vogel Osmometer OM815, was used for osmolality measurements        (calibrated with 400 mOsmol/kg NaCl calibration solution, Art.        No. Y1241, Herbert Knauer GmbH, Berlin, Germany).    -   Anton Paar Microviscosimeter, type AWVn, was used for viscosity        assessment of the protein solutions according to Anton Paar        Operating Instructions. Viscosity was assessed at 20° C.    -   Fluostar Optima, BMG Labtech (absorption measurement at 344 nm        in well plates, assessment of turbidity)    -   Eppendorf Centrifuge 5810 R    -   Slide-A-Lyzer dialysis cassettes, Pierce Biotechnology (Cat No        66830)    -   Vivaspin 20 concentrators, 10 KDa PES membranes (Vivascience,        Product number VS2001), used according to standard Operating        Instructions    -   PerkinElmer UV visible spectrophotometer, Lambda 35, was used        for protein concentration measurements (280 nm wavelength).        Disposable UV cuvettes, 1.5 mL, semi-micro, Poly(methyl        methacrylate) (PMMA), were used for the concentration        measurements.    -   An InoLab Cond Level2 WTW device was used for conductivity        measurements normalized to 25° C.    -   A Malvern Instruments Zetasizer nano ZS, was used for        determination of Z-average values, applying a standard method.        Measurements were performed at 25° C., using viscosity data        obtained by falling ball viscosimetry (Anton Paar        Microviscosimeter, type AWVn, at 25° C.).        HPLC Methods    -   J695, SEC analysis: Tosoh Bioscience G3000swxl, 7.8 mm×30 cm, 5        (Cat. No. 08541). Mobile phase 211 mM Na₂SO₄/92 mM Na₂HPO₄, pH        7.0. 0.25 mL/min flow rate, ambient temperature, detection UV        214 nm and 280 nm. Each sample was diluted to 2.0 mg/mL with        Milli-Q water, sample injection load 20 μg (duplicate        injection).    -   J695, IEC analysis: Dionex, Propac WCX-10 column (p/n 054993)        along with a corresponding guard column (p/n 054994). Separation        conditions: mobile phase A: 10 mM Na₂HPO₄, pH 6.0; mobile phase        B 10 mM Na₂HPO₄, 500 mM NaCl, pH 6.0. 1.0 mL/min flow rate,        35° C. temperature. Each sample was diluted to 1.0 mg/mL with        Milli-Q water, sample injection load 100 μg.        Calculation of the Protein Concentration

Calculation Formula:

$E = {{{- l}{g\left( \frac{I}{I_{0}} \right)}} = {\left. {ɛ \cdot c \cdot d}\rightarrow c \right. = \frac{E}{ɛ \times d}}}$ɛ − absorption  coefficientc − concentrationd − length  of  cuvette  that  the  light  has  to  passE − absorbanceI₀ − initial  light  intensityI − light  intensity  after  passing  through  sample$ɛ_{Adalimumab} = {1.42\frac{mL}{{mg} \times {cm}}}$Processing of J695

J695 in water was characterized prior to the addition of any non-ionicexcipients. Table 57 provides details of the J695 characterizationduring dialysis/ultrafiltration.

TABLE 57 Protein PCS Concentration Osmolality Viscosity VisualConductivity [Z-average/ Sample [mg/mL] PH [mosmol/kg] [cP] Inspection[μS/cm] d · nm] Starting ~125 mg/mL 6.29 (for N/A N/A Slightly N/A N/Amaterial low pH opalescent, samples, essentially adjusted to free from4.77 with visible 0.01M particles hydrochloric acid) After dialysis,42.5 mg/mL 5.21 7 1.60 Slightly 602 1.5 low pH opalescent, essentiallyfree from visible particles After dialysis, 56.9 mg/mL 6.30 6 2.11Slightly 500 2.7 high pH opalescent, essentially free from visibleparticles After 206 mg/mL 5.40 50 39.35 Slightly 1676 0.21concentration, opalescent, low pH essentially free from visibleparticles After 182 mg/mL 6.46 39 47.76 Slightly 1088 0.21concentration, opalescent, high pH essentially free from visibleparticlesCharacterization of Formulations with Non-Ionic Excipients

Following the addition of the various non-ionic excipients to the J695formulation (see description in Table 56), each formulation wasanalysed. Results from osmolality and visual inspection, and pH aredescribed below in Table 58.

TABLE 58 Osmolality Sample pH [mosmol/kg] Visual Inspection LI200/01/55.39 473 Slightly opalescent, essentially free from visible particlesLI200/02/5 5.38 402 Slightly opalescent, essentially free from visibleparticles LI200/03/5 5.37 466 Slightly opalescent, essentially free fromvisible particles LI200/04/5 5.37 397 Slightly opalescent, essentiallyfree from visible particles LI200/05/5 5.37 458 Slightly opalescent,essentially free from visible particles LI200/06/5 5.37 396 Slightlyopalescent, essentially free from visible particles LI200/07/5 5.36 50Slightly opalescent, essentially free from visible particles LI200/08/55.36 48 Slightly opalescent, essentially free from visible particlesLI200/01/6 6.43 428 Slightly opalescent, essentially free from visibleparticles LI200/02/6 6.42 405 Slightly opalescent, essentially free fromvisible particles LI200/03/6 6.43 348 Slightly opalescent, essentiallyfree from visible particles LI200/04/6 6.43 383 Slightly opalescent,essentially free from visible particles LI200/05/6 6.42 432 Slightlyopalescent, essentially free from visible particles LI200/06/6 6.42 402Slightly opalescent, essentially free from visible particles LI200/07/66.43 38 Slightly opalescent, essentially free from visible particlesLI200/08/6 6.43 39 Slightly opalescent, essentially free from visibleparticlesHPLC Data

Each of the non-ionic excipient containing J695 formulations were alsoexamined using SE-HPLC and IEX. The data from these analyses areprovided in Tables 59 and 60 and provide an overview of J695 stabilityduring processing and formulation.

TABLE 59 SE-HPLC results of various J695 formulations Sum aggregatesMonomer Sum Fragments sample name [%] [%] [%] pH 5 0.608 98.619 0.773 125 mg/mL 0.619 98.598 0.783 starting mat. 0.614 98.608 0.778 pH 60.427 98.809 0.764  125 mg/mL 0.392 99.005 0.603 starting mat. 0.40998.907 0.683 pH 5 0.654 98.604 0.742 42.5 mg/mL 0.677 98.560 0.763 afterdialysis 0.666 98.582 0.753 pH 6 0.748 98.541 0.711 56.9 mg/mL 0.73998.597 0.665 after dialysis 0.743 98.569 0.688 pH 5 0.913 98.416 0.671 206 mg/mL 0.923 98.356 0.721 In Water 0.918 98.386 0.696 pH 5 0.92898.312 0.760 50 mg/mL mannitol 0.926 98.339 0.736 LI 200/01/5 0.92798.325 0.748 pH 5 0.925 98.319 0.755 80 mg/mL sucrose 0.929 98.332 0.738LI 200/02/5 0.927 98.326 0.747 pH 5, 50 mg mannitol 0.942 98.326 0.7320.01% Tween 80 0.942 98.300 0.758 LI 200/03/5 0.942 98.313 0.745 pH 5,80 mg sucrose 0.944 98.315 0.741 0.01% Tween 80 0.944 98.339 0.717 LI200/04/5 0.944 98.327 0.729 pH 5, 50 mg mannitol 0.941 98.348 0.711 0.1% Tween 80 9.967 98.299 0.734 LI 200/05/5 0.954 98.323 0.722 pH 5,50 mg mannitol 0.944 98.346 0.710  0.1% Tween 80 0.948 98.340 0.712 LI200/06/5 0.946 98.343 0.711 pH 5, 50 mg mannitol 0.946 98.348 0.706 0.1% Tween 80 0.953 98.328 0.719 LI 200/07/5 0.949 98.338 0.713 pH 5,50 mg mannitol 0.987 98.313 0.701  0.1% Tween 80 0.994 98.283 0.723 LI200/08/5 0.991 98.298 0.712 pH 6 1.091 98.169 0.739  182 mg/mL 1.07598.221 0.703 In Water 1.083 98.195 0.721 pH 6 0.998 98.350 0.652 50mg/mL mannitol 1.002 98.364 0.634 LI 200/01/6 1.000 98.357 0.643 pH 61.028 98.243 0.729 80 mg/mL sucrose 0.983 98.355 0.662 LI 200/02/6 1.00698.299 0.695 pH 6, 50 mg mannitol 1.005 98.322 0.673 0.01% Tween 801.008 98.317 0.676 LI 200/03/6 1.006 98.319 0.674 pH 6, 80 mg sucrose0.987 98.363 0.649 0.01% Tween 80 0.987 98.321 0.692 LI 200/04/6 0.98798.342 0.671 pH 6, 50 mg mannitol 0.996 98.326 0.678  0.1% Tween 800.996 98.338 0.666 LI 200/05/6 0.996 98.332 0.672 pH 6, 80 mg sucrose0.998 98.305 0.697  0.1% Tween 80 0.984 98.345 0.671 LI 200/06/6 0.99198.325 0.684 pH 6 1.000 98.325 0.675 0.01% Tween 80 0.994 98.347 0.659LI 200/07/6 0.997 98.336 0.667 pH 6 1.003 98.314 0.682 0.01% Tween 800.998 98.338 0.664 LI 200/08/6 1.001 98.326 0.673

TABLE 60 LEX results of various J695 formulations Sum Acid Peaks SumGlutamine Sum Basic Peaks sample name [%] [%] [%] pH 5 4,598 3,303  125mg/mL 4,599 3,177 starting mat. 4,599 92,162 3,240 pH 6 4,597 3,159  125mg/mL 4,629 3,156 starting mat. 4,613 92,229 3,158 pH 5 4,706 3,177 42.5mg/mL 4,725 3,205 after dialysis 4,715 92,094 3,191 pH 6 4,739 3,18256.9 mg/mL 4,752 3,167 after dialysis 4,746 92,080 3,174 pH 5 4,6553,167  206 mg/mL 4,676 3,201 In Water 4,666 92,146 3,189 pH 5 4,7213,321 50 mg/mL mannitol 4,733 3,356 LI 200/01/5 4,727 92,935 3,338 pH 54,715 3,299 80 mg/mL sucrose 4,687 3,338 LI 200/02/5 4,701 92,981 3,318pH 5, 50 mg mannitol 4,767 3,246 0.01% Tween 80 4,736 3,253 LI 200/03/54,752 92,999 3,250 pH 5, 80 mg sucrose 4,751 3,257 0.01% Tween 80 4,7423,229 LI 200/04/5 4,746 92,011 3,243 pH 5, 50 mg mannitol 4,780 3,420 0.1% Tween 80 4,720 3,394 LI 200/05/5 4,750 92,843 3,407 pH 5, 50 mgmannitol 4,756 3,421  0.1% Tween 80 4,894 3,375 LI 200/06/5 4,825 92,7773,398 pH 5, 50 mg mannitol 4,813 3,425  0.1% Tween 80 4,757 3,413 LI200/07/5 4,785 92,796 3,419 pH 5, 50 mg mannitol 4,769 3,361  0.1% Tween80 4,842 3,335 LI 200/08/5 4,806 92,846 3,348 pH 6 4,882 3,452  182mg/mL 4,886 3,451 In Water 4,884 92,664 3,451 pH 6 4,843 3,456 50 mg/mLmannitol 4,833 3,393 LI 200/01/6 4,838 92,737 3,425 pH 6 4,923 3,407 80mg/mL sucrose 4,896 3,491 LI 200/02/6 4,909 92,642 3,449 pH 6, 50 mgmannitol 4,864 3,423 0.01% Tween 80 4,899 3,392 LI 200/03/6 4,882 92,7113,408 pH 6, 80 mg sucrose 4,870 3,320 0.01% Tween 80 4,928 3,369 LI200/04/6 4,899 92,756 3,345 pH 6, 50 mg mannitol 4,905 3,385  0.1% Tween80 4,922 3,489 LI 200/05/6 4,914 92,649 3,437 pH 6, 80 mg sucrose 4,9733,443  0.1% Tween 80 4,962 3,335 LI 200/06/6 4,968 92,644 3,389 pH 64,934 3,413 0.01% Tween 80 4,899 3,392 LI 200/07/6 4,916 92,681 3,402 pH6 4,884 3,410 0.01% Tween 80 4,934 3,366 LI 200/08/6 4,909 92,703 3,388Conclusion

The above example provides an experiment where water (demineralised andsterile filtered water) was used as dialysis medium for the monoclonalantibody J695.

J695 was subjected to dialysis and concentration processing by usingpure water as exchange medium and was formulated at pH 5.40 as well as6.46 at high concentration (206 and 182 mg/mL, respectively) withoutinducing solution haziness, severe opalescence or turbidity formation.

J695 from the processing experiment was characterized, and formulatedwith various non-ionic excipients. Data obtained points at favorableoverall stability of the protein in the formulations tested.

In conclusion, processing proteins using pure water as exchange mediumand formulation with non-ionic excipients is feasible. Assuming an ideal100% excipient membrane permeability, an approx. 99.9% maximum excipientreduction can be estimated.

Example 24: Syringeability of Adalimumab Formulated in Water

The formulations from example 21 (63 and 220 mg/mL Adalimumab) weresubjected to force measurements upon syringe depletion. 220 mg/mLsamples of Adalimumab were diluted to 200 mg/mL, 150 mg/mL and 100mg/mL, respectively, and were also assessed. A Zwick Z2.5/TN1S was usedat a constant feed of 80 mm/min. Finally, viscosity data of theformulations was assessed using an Anton Paar Microviscosimeter, typeAWVn, at 20° C. The following data collection suggests that both needleand syringe diameters have a significant effect on the gliding forcesupon syringe depletion. Surprisingly, the highly concentrated solutionat 220 mg/mL (viscosity 27.9 cP at 20° C.) can be delivered by applyingequivalent depletion forces as with the lower concentrated formulationat 63 mg/mL (viscosity 1.8 cP at 20° C.).

TABLE 61 Gliding Force Values obtained for Adalimumab solutions indifferent packaging systems. D HyPak SCF ™ 1 mL long syringes, equippedwith 25 G × 26 G × 27 G × BD HyPak ⅝″ ½″ ½″ SCF ™ 1 mL long needlesneedles needles 1 mL Soft-Ject ® syringes, (Sterican) (Sterican)(Sterican) Tuberkulin syringes equipped with BD BD BD (smaller diameter27.5 G RNS HyPak HyPak HyPak than BD HyPak Adaliumab needles BSCF BSCFBSCF syringes), equipped Concentration BD HyPak BSCF 4432/50 4432/504432/50 with 27 G × ½″ (Viscosity) 4432/50 stoppers stoppers stoppersstoppers needles (Sterican)  63 mg/mL  3.9N — — — — (1.8 cP) 100 mg/mL3.30N 1.02N 1.33N 1.69N 1.00N (2.9 cP) 150 mg/mL 4.63N 1.16N 1.58N 2.93N1.33N (7.4 cP) 200 mg/mL 7.25N 2.16N 3.24N 6.25N 2.55N (15.7 cP)  220mg/mL 14.5N 2.99N 3.97N 9.96N 3.16N (27.9 cP) 

The above suggests that even with high concentrations of protein, suchformulations are conducive to administration using a syringe, e.g.,subcutaneous.

Examples 25-28

Examples 25-28 describe freeze/thaw stability experiments of variousantibody formulations containing the antibody formulated in water(referred to in examples 26-28 as low-ionic strength proteinformulations). The freeze thaw behavior of a number of antibodies wasevaluated by cycling various protein formulations up to 4 times betweenthe frozen state and the liquid state. Freezing was performed by meansof a temperature controlled −80° C. freezer, and thawing was performedby means of a 25° C. temperature controlled water bath. About 25 mL ofantibody solution each were filled in 30 mL PETG repositories for theseexperiment series.

Formation of subvisible particles presents a major safety concern inpharmaceutical protein formulations. Subvisible protein particles arethought to have the potential to negatively impact clinical performanceto a similar or greater degree than other degradation products, such assoluble aggregates and chemically modified species that are evaluatedand quantified as part of product characterization and quality assuranceprograms (Carpenter, J F et al. Commentary: Overlooking subvisibleparticles in therapeutic protein products: baps that may compromiseproduct quality. J. Pharm. Sci., 2008). As demonstrated in the exampleslisted below, a number of antibodies were surprisingly stable—especiallywith regard to subvisible particle formation—when formulated in theformulation of invention.

Example 25: Freeze/Thaw Stability of Adalimumab Formulated in Water andwith Non-Ionic Excipients

The following example describes the stability of an antibody, e.g.,adalimumab, in a water formulation and in water formulations in whichnon-ionic excipients have been added. Aliquots of samples from examples21 and 22 were subjected to freeze/thaw experiments and analyzed bySE-HPLC. Data was compared to SE-HPLC results derived from freeze/thawexperiments using Adalimumab in a buffer of the following composition:10 mM phosphate buffer, 100 mM sodium chloride, 10 mM citrate buffer, 12mg/mL mannitol, 0.1% polysorbate 80, pH 5.2. Adalimumab in this latterbuffer was used at 50 mg/mL and 200 mg/mL, respectively. Freeze/thawcycles were performed in Eppendorf caps, by freezing to −80° C. andstorage in the freezer for 8 hours, followed by thawing at roomtemperature for 1 hour and subsequent sample pull. Each formulation wassubjected to 5 cycles, i.e., cycles 0, 1, 2, 3, 4, and 5 described inthe tables below.

HPLC Method

Adalimumab, SEC analysis: Sephadex 200 column (Pharmacia Cat. No.175175-01). Mobile phase 20 mM sodium phosphate, 150 mM sodium chloride,pH 7.5, 0.5 mL/min flow rate, ambient temperature, detection UV 214 nmand 280 nm. Each sample was diluted to 1.0 mg/mL with Milli-Q water,sample injection load 50 μg (duplicate injection).

Adalimumab Characterization Upon Freeze/Thaw Cycling

Table 62 describes Adalimumab purity during the freeze/thaw experiments.For sample composition, refer to examples 21 and 22.

TABLE 62 Freeze/thaw-Low ionic Adalimumab-50 mg/mL Fraction monomer [%]cycle LI 50/01 LI 50/02 LI 50/03 LI 50/04 LI 50/05 LI 50/06 LI 50/07 LI50/08 0 99.605 99.629 99.632 99.626 99.619 99.626 99.653 99.655 1 99.74399.768 99.739 99.759 99.731 99.725 99.686 99.669 2 99.715 99.726 99.57199.661 99.721 99.721 99.601 99.619 4 99.668 99.689 99.632 99.678 99.52399.724 99.491 99.542 5 99.627 99.771 99.539 99.772 99.525 99.773 99.35799.445 Fraction aggregates [%] cycle LI 50/01 LI 50/02 LI 50/03 LI 50/04LI 50/05 LI 50/06 LI 50/07 LI 50/08 0 0.106 0.104 0.119 0.136 0.1390.145 0.158 0.159 1 0.149 0.134 0.153 0.150 0.149 0.166 0.206 0.201 20.192 0.178 0.320 0.242 0.178 0.184 0.319 0.261 4 0.213 0.185 0.2390.187 0.357 0.170 0.393 0.353 5 0.301 0.151 0.384 0.150 0.398 0.1500.568 0.484 Fraction fragments [%] cycle LI 50/01 LI 50/02 LI 50/03 LI50/04 LI 50/05 LI 50/06 LI 50/07 LI 50/08 0 0.289 0.267 0.249 0.2380.242 0.229 0.189 0.186 1 0.108 0.098 0.108 0.091 0.120 0.108 0.1070.130 2 0.093 0.097 0.108 0.097 0.100 0.094 0.080 0.119 4 0.119 0.1260.130 0.135 0.120 0.106 0.116 0.105 5 0.072 0.078 0.078 0.078 0.0770.077 0.075 0.071 Freeze/thaw-Low ionic Adalimumab-200 mg/mL Fractionmonomer [%] cycle LI 200/01 LI 200/ 02 LI 200/03 LI 200/04 LI 200/05 LI200/06 LI 200/07 LI 200/08 0 99.294 99.296 99.348 99.333 99.313 99.34999.320 99.290 1 99.286 99.267 99.259 99.256 99.120 99.254 98.999 99.1262 99.305 99.311 99.249 99.214 99.296 99.288 99.149 99.128 4 99.30399.272 99.261 99.301 99.296 99.283 99.004 99.061 5 99.320 99.322 99.33099.331 99.327 99.333 98.939 98.949 Fraction aggregates [%] cycle LI200/01 LI 200/02 LI 200/03 LI 200/04 LI 200/05 LI 200/06 LI 200/07 LI200/08 0 0.489 0.509 0.491 0.484 0.492 0.488 0.515 0.575 1 0.590 0.5740.584 0.586 0.680 0.582 0.785 0.718 2 0.604 0.604 0.616 0.630 0.6070.607 0.731 0.736 4 0.591 0.592 0.612 0.581 0.604 0.596 0.868 0.836 50.593 0.586 0.583 0.596 0.597 0.589 0.985 0.981 Fraction fragments [%]cycle LI 200/01 LI 200/02 LI 200/03 LI 200/04 LI 200/05 LI 200/06 LI200/07 LI 200/08 0 0.218 0.196 0.161 0.183 0.195 0.163 0.165 0.135 10.124 0.159 0.157 0.159 0.200 0.164 0.216 0.157 2 0.091 0.085 0.1350.156 0.097 0.105 0.120 0.136 4 0.106 0.136 0.127 0.118 0.100 0.1210.128 0.103 5 0.087 0.092 0.087 0.073 0.075 0.078 0.076 0.070Freeze/thaw-Adalimumab Commercial and in water Fraction monomer [%] fromexample A, from example A, Standard, Standard, cycle low conc. highconc. 50 mg/mL 200 mg/mL 0 99.733 99.286 99.374 99.227 1 99.689 99.21299.375 99.215 2 99.614 99.130 99.370 99.218 4 99.489 99.029 99.36199.196 5 99.430 98.945 99.362 99.177 Fraction aggregates [%] fromexample A, from example A, Standard, Standard, cycle low conc. highconc. 50 mg/mL 200 mg/mL 0 0.186 0.635 0.358 0.502 1 0.226 0.706 0.3590.516 2 0.304 0.780 0.364 0.513 4 0.428 0.888 0.372 0.535 5 0.485 0.9710.373 0.553 Fraction fragments [%] from example A, from example A,Standard, Standard, cycle low conc. high conc. 50 mg/mL 200 mg/mL 00.080 0.079 0.268 0.272 1 0.085 0.083 0.266 0.269 2 0.082 0.090 0.2660.269 4 0.083 0.083 0.267 0.270 5 0.085 0.085 0.265 0.269Conclusion

The above example provides an experiment where Adalimumab DF/UFprocessed into water (Sterilized water for injection Ph.Eur./USP) andformulated with various non-ionic excipients was subjected tofreeze/thaw cycling. Data obtained (described in Table 62) indicatesfavorable overall stability of the protein in all formulations tested.All formulations contained above 98.5% monomeric species after 5freeze/thaw cycles, with minimal amounts of aggregate or fragments ascycles continued.

Example 26: Freeze/Thaw Stability of Low-Ionic 1D4.7 Solutions

1D4.7 protein (an anti-IL 12/anti-IL 23 IgG1) was formulated in water bydialysis (using slide-a-lyzer cassettes, used according to operatinginstructions of the manufacturer, Pierce, Rockford, Ill.) and wasdemonstrated to be stable during repeated freeze/thaw (f/t) processing(−80° C./25° C. water bath) at 2 mg/mL concentration, pH 6. Data werecompared with data of various formulations (2 mg/mL protein, pH 6) usingbuffers and excipients commonly used in parenteral protein formulationdevelopment. It was found that the stability of 1D4.7 formulated inwater exceeded the stability of 1D4.7 formulated in established buffersystems (e.g. 20 mM histidine, 20 mM glycine, 10 mM phosphate, or 10 mMcitrate) and even exceeded the stability of 1D4.7 formulations based onuniversal buffer (10 mM phosphate, 10 mM citrate) combined with avariety of excipients that are commonly used to stabilize proteinformulations, e.g. 10 mg/mL mannitol, 10 mg/mL sorbitol, 10 mg/mLsucrose, 0.01% polysorbate 80, or 20 mM NaCl.

SEC, DLS and particle counting analysis were applied to monitor proteinstability, and particle counting was performed using a particle countingsystem with a 1-200 μm measurement range (particle counter ModelSyringe, Markus Klotz GmbH, Bad Liebenzell, Germany). Experiment detailsare as follows:

-   -   1D4.7 formulated in water compared with formulations listed        above    -   4 freeze/thaw cycles applied    -   30 mL PETG repository, about 20 mL fill, 2 mg/mL protein, pH 6    -   sampling at T0, T1 (i.e. after one f/t step), T2, T3, and T4    -   analytics: visual inspection, SEC, DLS, subvisible particle        measurement

FIG. 33 shows 1D4.7 stability during repeated f/t cycling (−80° C./25°C.), mirrored by formation of subvisible particles >1 μm. 1D4.7 wasformulated in universal buffer (10 mM citrate, 10 mM phosphate) and thenthe following excipient variations were tested: sorbitol (10 mg/mL),mannitol (10 mg/mL), sucrose (10 mg/mL), NaCl (100 mM), and polysorbate80 (0.01%). 1D4.7 was also formulated in water (by dialysis) with noexcipients added at all (“water” in FIG. 33). Water for injection wasalso subjected to f/t cycling and subvisible particle testing toevaluate a potential impact of material handling, f/t, and sample pullon particle load.

The stability of 1D4.7 formulated in water upon f/t exceeded thestability of 1D4.7 solutions formulated with excipients typically usedin protein formulations. Mannitol, sucrose, and sorbitol are known toact as lyoprotectant and/or cryoprotectant, and polysorbate 80 is anon-ionic excipient prevalently known to increase physical stability ofproteins upon exposure to hydrophobic-hydrophilic interfaces such asair-water and ice-water, respectively.

In summary, 1D4.7 solutions formulated in water appeared to besurprisingly stable when analyzed with various analytical methodologiestypically applied to monitor stability of pharmaceutical proteins uponfreeze-thaw processing (e.g. SEC, visual inspection, dynamic lightscattering, and especially light obscuration).

Example 27: Freeze/Thaw Stability of Low-Ionic 13C5.5 Solutions

13C5.5 (an anti IL-13 IgG1) formulated in water was demonstrated to bestable during repeated freeze/thaw processing (−80° C./25° C. waterbath) at 2 mg/mL concentration, pH 6. Data were compared with otherformulations (2 mg/mL protein, pH 6), and it was found that thestability of 13C5.5 formulated in water exceeded the stability of 13C5.5formulated in buffer systems often used in parenteral proteinformulations (e.g. 20 mM histidine, 20 mM glycine, 10 mM phosphate, or10 mM citrate) and even exceeded the stability of 13C5.5 formulationsbased on universal buffer (10 mM phosphate, 10 mM citrate) that has beencombined with a variety of excipients that are commonly used in proteinformulation (e.g. 10 mg/mL mannitol, 10 mg/mL sorbitol, 10 mg/mLsucrose, 0.01% polysorbate 80, 20 mM NaCl, 200 mM NaCl).

Sample preparation, experiment processing, sample pull and sampleanalysis was performed in the same way as outlined in the aboveexamples.

-   -   13C5.5 formulated in water compared with formulations listed        above    -   4 freeze/thaw cycles applied    -   30 mL PETG repository    -   2 mg/mL, pH 6    -   sampling at T0, T1, T2, T3, and T4    -   analytics: visual inspection, SEC, DLS, subvisible particle        measurement

FIG. 34 shows 13C5.5 stability during repeated f/t cycling (−80° C./25°C.), mirrored by formation of subvisible particles >10 μm. 13C5.5 wasformulated in either 10 mM phosphate buffer, 10 mM citrate buffer, 20 mMglycine buffer, and 20 mM histidine buffer. 13C5.5 was also formulatedin the formulation of invention (by dialysis) with no excipients addedat all. Water for injection was also subjected to f/t cycling andsubvisible particle testing to evaluate a potential impact of materialhandling, f/t, and sample pull on particle load (referred to as blank).

The stability of 13C5.5 formulated in water upon f/t exceeded thestability of 13C5.5 solutions formulated in buffers typically used inprotein formulations. No instabilities of 13C5.5 solutions formulated inwater have been observed with other analytical methodologies applied(e.g. SEC, visual inspection, etc.)

FIG. 35 shows 13C5.5 stability during repeated f/t cycling (−80° C./25°C.), mirrored by formation of subvisible particles >1 μm. 13C5.5 wasformulated in universal buffer (10 mM citrate, 10 mM phosphate) and inuniversal buffer combined with the following excipient variations weretested: sorbitol (10 mg/mL), mannitol (10 mg/mL), sucrose (10 mg/mL),NaCl (200 mM), NaCl (20 mM) and polysorbate 80 (0.01%). 13C5.5 was alsoformulated in water (by dialysis) with no excipients added at all forcomparison (pure water). Water for injection was also subjected to f/tcycling and subvisible particle testing to evaluate a potential impactof material handling, f/t, and sample pull on particle load.

The stability of 13C5.5 formulated in water upon f/t exceeded thestability of 13C5.5 solutions formulated with excipients typically usedin protein formulations. Mannitol, sucrose, and sorbitol are known toact as lyoprotectant and/or cryoprotectant, and polysorbate 80 is anon-ionic excipient prevalently known to increase physical stability ofproteins upon exposure to hydrophobic-hydrophilic interfaces such asair-water and ice-water, respectively. The low number of subvisibleparticles in 13C5.5 samples formulated into the formulation of inventionwas found to be at surprisingly low levels, demonstrating the highsafety and stability potential of such formulations.

No instabilities of 13C5.5 solutions formulated in water have beenobserved with other analytical methodologies applied, (e.g. SEC, visualinspection, etc.).

DLS analysis of 13C5.5 solutions after f/t procedures was performed asdescribed above. Results from the DLS analysis showed that an 13C5.5solution with 0.01% Tween-80 contained significant high molecular weight(HMW) aggregate forms after only 1 f/t step, whereas 13C5.5 in watercontained no BMW aggregate forms, even after 3 f/t steps applied.

In summary, 13C5.5 solutions formulated in water appeared to besurprisingly stable when analyzed with various analytical methodologiestypically applied to monitor stability of pharmaceutical proteins uponfreeze-thaw processing (e.g. SEC, visual inspection, dynamic lightscattering, and especially light obscuration).

Example 28: Freeze/Thaw Stability of Low-Ionic 7C6 Solutions

7C6 (an anti amyloid beta IgG1) formulated in water was demonstrated tobe stable during repeated freeze/thaw processing (−80° C./30° C. waterbath) at 2 mg/mL concentration, pH 6. Data were compared with otherformulations (2 mg/mL protein, pH 6), and it was found that thestability of 7C6 formulated in water exceeded the stability of 7C6formulated in buffer systems often used in parenteral proteinformulations and even exceeded the stability of 7C6 formulations basedon universal buffer (10 mM phosphate, 10 mM citrate) that has beencombined with a variety of excipients that are commonly used in proteinformulation.

The following solution compositions were evaluated for their potentialto maintain 7C6 physical stability during freeze/thaw experiments:

-   -   Phosphate buffer, 15 mM    -   Citrate buffer, 15 mM    -   Succinate buffer, 15 mM    -   Histidine buffer, 15 mM    -   Arginine buffer, 15 mM    -   Low ionic protein formulation, no excipients added    -   Universal buffer, sorbitol (10 mg/mL)    -   Universal buffer, mannitol (10 mg/mL)    -   Universal buffer, sucrose (10 mg/mL)    -   Universal buffer, trehalose (10 mg/mL)    -   Universal buffer, 0.01% (w/w) polysorbate 80

Sample preparation, experiment processing, sample pull and sampleanalysis was performed in very similar way as outlined in Examples 26and 27.

-   -   7C6 formulated in water compared with formulations listed above    -   4 freeze/thaw cycles applied    -   30 mL PETG repository, approx. 20 mL fill    -   2 mg/mL, pH 6    -   sampling at T0, T1, T2, T3, and T4    -   Analytics: Aβ-antibody stability was assessed by the following        methods:    -   Visual inspection of the protein solution was performed in        polypropylene round-bottom tubes wherein the samples were filled        for light obscuration measurements.

It was carefully inspected against both a black and a white backgroundfor signs indicating protein physical instability such as haziness,turbidity and particle formation.

-   -   Dynamic light scattering (eZetasizer Nano ZS, Malvern        Instruments, AI9494;    -   equipped with Hellma precision cells, suprasil quartz, type        105.251-QS, light path 3 mm, center Z8.5 mm, at least 60 μL        sample fill, protein sample remaining from light obscuration        measurements in PP round-bottom tubes were used for DLS        measurements). Automated measurements (1 measurement per sample)        were performed.    -   Light obscuration analysis. 3.5 mL of sample were filled in 5 mL        round-bottom tube under laminar air flow conditions, measurement        was performed in n=3 mode (0.8 mL per single measurement) after        an initial 0.8 mL rinse.    -   Size-exclusion chromatography, combined with UV₂₁₄/UV₂₈₀ and        multi-angle light scattering. Mobile phase: 100 mM Na2HPO4/200        mM Na2SO4, pH 7.0 (49.68 g anhydrous disodium hydrogen phosphate        and 99.44 g anhydrous sodium sulfate were dissolved in approx.        3300 mL Milli-Q water, the pH was adjusted to 7.0 using 1 M        phosphoric acid, filled to a volume of 3500 mL with Milli-Q        water, and the solution was filtered through a membrane filter).        SEC column, TSK gel G3000SW (cat. no. 08541) 7.8 mm×30 cm, 5 μm        combined with a TSK gel guard (cat. no. 08543) 6.0 mm×4.0 cm, 7        μm. Flow 0.3 mL/min, injection volume 20 μL (equivalent to 20 μg        sample), column temperature room temperature, autosampler temp.        2 to 8° C., run time 50 minutes, gradient isocratic, piston        rinsing with 10% isopropyl alcohol, detection via UV absorbance,        diode array detector: wavelength 214 nm, peak width >0.1 min,        band width: 8 nm, reference wavelength 360 nm, band width 100 nm

FIG. 36 shows 7C6 stability during repeated f/t cycling (−80° C./25°C.), mirrored by formation of subvisible particles >1 μm. The stabilityof 7C6 formulated in water upon f/t for many formulations exceeded thestability of 7C6 solutions formulated in buffers typically used inprotein formulations. No instabilities of 7C6 solutions formulated inwater have been observed with other analytical methodologies applied(e.g. SEC, visual inspection, dynamic light scattering)

Surprisingly, the stability of 7C6 formulated in water upon f/t exceededthe stability of 7C6 solutions formulated with excipients typically usedin protein formulations. Mannitol, sucrose, and sorbitol are known toact as lyoprotectant and/or cryoprotectant, and polysorbate 80 is anon-ionic excipient prevalently known to increase physical stability ofproteins upon exposure to hydrophobic-hydrophilic interfaces such asair-water and ice-water, respectively. The low number of subvisibleparticles in 7C6 samples formulated into the formulation of inventionwas found to be at surprisingly low levels, demonstrating the highsafety and stability potential of such formulations.

In summary, 7C6 solutions formulated in water appeared to besurprisingly stable when analyzed with various analytical methodologiestypically applied to monitor stability of pharmaceutical proteins uponfreeze-thaw processing (e.g. SEC, visual inspection, dynamic lightscattering, and especially light obscuration).

Example 29: Preparation of J695 Formulated in Water and StabilityStudies Thereof

Materials and Methods

427.1 g (80 mg/mL) of J695 were diluted to 40 mg/mL and diafilteredusing purified water. After a 5-fold volume exchange with purified water(theoretical excipients reduction, 99.3%), the protein solution wasultrafiltered to target concentration of 100 mg/mL. pH, osmolality,density, visual inspection and protein concentration measurements(OD280) were performed to monitor the status of the protein after DF/UFprocessing.

After DF/UF processing, the protein solution was sterile filtered (0.22μm Sterivex GV membrane filter) into a 60 mL PETG bottle (Nalgene) andsubsequently stored at −80° C. for 3 months.

After thawing at 37° C., the solution was sterile filtered (0.22 μmSterivex GV membrane filter) and filled into sterile BD Hypak PhysiolisSCF™ 1 mL long syringes 29 G, ½ inch, 5-bevel, RNS TPE and closed withsterile BD Hypak SCF™ 1 ml W4023/50 Flur Daikyo stoppers. The fillingvolume was 1.000 mL per syringe.

After filling, the syringes were stored at 2-8° C. and 40° C.,respectively, and analyzed as indicated in the sample pull schemedepicted below.

-   -   J695 Drug Substance (extinction coefficient at 280 nm: 1.42        mL/mg cm): Drug Substance, pH 6.0, did not contain polysorbate        80.    -   Sartorius Sartocon Slice diafiltration system, equipped with        Ultrasert PES membrane cassettes (50 kDa cutoff). The Sartocon        Slice system was operated in continuous mode at ambient        temperature according to Sartorius Operating Instructions.    -   pH electrodes    -   Perkin Elmer UV/Vis spectrophotometer, Lambda 25, was used for        protein concentration measurements (280 nm wavelength).        Disposable UV cuvettes, 1.5 mL, semi-micro, were used for the        concentration measurements.    -   0.22 μm filtered purified water was used as DF/UF medium.    -   Anton Paar Density Meter DMA 4100 was used for density        measurements    -   A Knauer Osmometer Type ML, was used for osmolality measurements        (calibrated with 400 mOsmol/kg NaCl calibration solution, Art.        No. Y1241, Herbert Knauer GmbH, Berlin, Germany).        Analytical Methods    -   J695, SEC analysis: Superdex 200 column (Pharmacia). Mobile        phase 92 mM di-sodium hydrogen phosphate, 211 mM sodium sulfate,        pH 7.0, 0.75 mL/min flow rate, ambient temperature, detection UV        214 nm. Each sample was diluted to 2.0 mg/mL with mobile phase,        sample injection load 20 μg (duplicate injection).    -   J695, IEC analysis: Dionex, Propac WCX-10 column along with a        corresponding guard column. Separation conditions: mobile phase        A: 20 mM di-sodium hydrogen phosphate and 20 mM sodium acetate,        pH 7.0; mobile phase B 20 mM di-sodium hydrogen phosphate, 400        mM Sodium chloride, pH 5.0. 1.0 mL/min flow rate, ambient        temperature. Each sample was diluted to 1.0 mg/mL with Milli-Q        water, sample injection load 100 μg (duplicate injection).    -   J695, SDS-PAGE analysis: Novex acryl amide slab gels (8-16% for        non-reducing conditions, 12% for reducing conditions,        Invitrogen), Coomassie staining (Invitrogen). Separation under        reducing (β-mercaptoethanol) and non-reducing conditions using        Tris-Glycine buffer made of 10× stock solution (Invitrogen).    -   J695, quantitation of buffer components:        -   Mannitol: separation per ReproGel Ca column (Dr. Maisch,            Germany) and RI detection, mobile phase: deionized water,            0.6 mL/min flow rate, 20 μL sample injection. Quantitation            was performed using external calibration standard curve.        -   Histidine and Methionine: fluorescence labelling of the            amino acids with OPA (ortho-phthalic aldehyde) and HPLC            separation per ReproSil ODS-3 column (Dr. Maisch, Germany)            and fluorescence detection at 420 nm (extinction at 330 nm),            mobile phase A: 70% citric acid (10.51 g/L) buffer, pH 6.5,            30% methanol, mobile phase B: methanol, 1.0 mL/min flow            rate, 20 μL sample injection. Quantitation was performed            using external calibration standard curve.    -   J695, PCS analysis: was performed undiluted at 100 mg/mL in        single-use plastic cuvettes at 25° C. using a A Malvern        Instruments Zetasizer nano ZS at 173° angle assuming solution        viscosity of 4.3875 mPas, refractive index of the protein of        1.450 and refractive index of the buffer solution of 1.335. The        averaged results of 20 scans, 20 seconds each, are reported.        Calculation of the Protein Concentration

Calculation Formula:

$E = {{- {\lg\left( \frac{I}{I_{0}} \right)}} = {\left. {ɛ \cdot c \cdot d}\rightarrow c \right. = \frac{E}{ɛ \times d}}}$ɛ − absorption  coefficient c − concentrationd − length  of  cuvette  that  the  light  has  to  pass E − absorbanceI₀ − initial  light  intensity I − light  intensity  after  passing$ɛ_{J\; 695} = {1.42\frac{mL}{{mg} \times {cm}}}$Sample Pull Scheme

Samples of the prepared solutions were stored at the temperatures listedbelow and pulled (x) at the indicated time points after study start(Table 63). Test parameters and methods are described in Table 64.

TABLE 63 Temp. T0 1 m 3 m 6 m  5° C. x x x x 40° C. x x x

TABLE 64 Test parameter Test method Appearance Visual inspection Visibleparticles analogous DAC (EA 4.43) Sub-visible particles analogous Ph.Eur./USP EA 4.44 Clarity Ph. Eur. (EA 4.42) Color (visual) Ph. Eur. (EA4.50) pH Ph. Eur. (EA 4.24) Size exclusion HPLC See above Cationexchange HPLC See above SDS-PAGE See above PCS See aboveDF/UF Processing of J695Table 65 provides the J695 status after diafiltration.

TABLE 65 Protein Concentration Osmolality Sample [mg/mL] pH [mosmol/kg]Visual Inspection after 107 6.4 10 Slightly opalescent, DF/UF slightlyyellow essentially free from visible particles

After DF/UF the concentrations of the originating buffer components werequantitatively monitored to assess the DF effectiveness. All resultswere found to be below the practical detection limits (see Table 66) ofthe corresponding analytical methods (HPLC with RI for Mannitol andfluorescence detection for the methionine and histidine after OPAlabeling, respectively).

TABLE 66 Methionine Histidine Mannitol Sample [mg/mL] [mg/mL] [mg/mL]before DF/UF 0.669 0.586 18.36 after DF/UF <0.13 <0.14 <3.20J695 Characterization upon storage Table 67 below supports the stabilityof J695 DF/UF at 100 mg/mL upon storage.

Duration of Storage conditions testing [° C./% RH] Test criteriaSpecification [months] +5 +40/75 Appearance solution Initial complies 1complies complies 3 complies complies 6 complies complies Clarity ReportResults, Initial ≤RSII Compare to reference suspensions acc. 1 — — toPh. Eur. 3 ≤RSII ≤RSII 6 ≤RSII ≤RSIII Particulate Report Result Initial2.0 (1) contamination Visual Score 1 2.0 (1) 3.0 (1) Visible particles(number of samples tested) 3 1.6 (5) 1.0 (5) 6 1.1 (9) 1.6 (9)Particulate ≥10 μm:≤6000 particles per initial 290 contaminationcontainer ≥10 μm 16 Subvisible particles ≥25 μm:≤600 particles percontainer ≥25 μm 1 — — ≥10 μm ≥25 μm 3 — — ≥10 μm ≥25 μm 6 124 54 ≥10 μm1 3 ≥25 μm Size Exclusion HPLC Report Results (%) for initial 0.9Aggregates (A) A 98.9 Monomer (M) M 0.2 Fragments (F) F 1 0.8 2.3 A 99.097.1 M 0.1 0.6 F 3 1.0 3.4 A 98.8 95.1 M 0.1 1.4 F 6 1.4 5.5 A 98.4 85.6M 0.1 8.9 F SDS-PAGE The predominant banding pattern is comparableInitial complies (Non-reducing to that of the reference standard. 1complies complies conditions) 3 complies complies 6 complies compliesSDS-PAGE The predominant banding pattern is comparable Initial complies(reducing to that of the reference standard. 1 complies compliesconditions) 3 complies complies 6 complies complies PCS Report Resultsfor initial 0.9 Z-Average [nm] and 0.23 PDI 1 0.9 1.0 0.23 0.23 3 0.91.1 0.23 0.24 6 0.9 1.3 0.23 0.29 Cation Exchange Report Results (%) forinitial 5.9 HPLC Acidic Species (A) A 91.5 Main Isoforms (M) M 2.5 BasicSpecies (B) B 1 5.6 10.6 A 92.0 98.9 M 2.4 0.5 B 3 5.7 14.4 A 92.1 85.0M 2.1 0.6 B 6 6.0 29.6 A 91.6 69.4 M 2.3 1.0 BConclusion

The above example provides a diafiltration/ultrafiltration (DF/UF)experiment where water (0.22 μm filtered purified water) was used asdiafiltration medium for the monoclonal antibody J695.

J695 was subjected to DF/UF processing using pure water as DF/UFexchange medium and was formulated at about pH 6.4 at high concentration(100 mg/mL) without inducing solution haziness, severe opalescence orturbidity formation.

J695 from the DF/UF experiments was stored in SCF syringes at 2-8° C.and 40° C. for up to 6 months. Data obtained points to a favourableoverall stability of the protein.

In conclusion, processing and formulating proteins using pure water asDF/UF exchange medium is feasible. Assuming an ideal 100% excipientmembrane permeability, an approx. 99.3% maximum excipient reduction canbe estimated. Evidence is given by specific methods that after DF/UF theexcipient concentration is below the practical detection limits.

Example 30: Freeze/Thaw Characteristics and Stability Testing of HighConcentration Adalimumab Water Solution—Homogeneity and PhysicalStability

Preparation of Low-Ionic Adalimumab Solutions

1.6 L of Drug Substance (DS) material in 2 L PETG bottle was thawed at25° C. in a water bath, homogenized and subjected to DF/UF using waterfor injection as a diafiltration exchange medium. Diafiltration wasperformed in continuous mode with Sartorius Sartocon Slice equipment byapplying the following parameter:

-   -   Pump output: 8%    -   Pressure inlet: max 1 bar (0.8 bar)    -   Membrane: 2×PES, cut off 50 kD

During the diafiltration 5-fold volume exchange was sufficient to reduceosmolality to 8 mOsmol/kg.

In-Process-Control (IPC) samples were pulled prior to diafiltration(SEC, protein concentration by means of OD280, pH, osmolality anddensity) and after diafiltration (protein concentration by means ofOD280, pH, osmolality and density). The IPC-samples were not sterile.

After diafiltration the ˜70 mg/mL Adalimumab formulated in water wasdiluted to 50 mg/mL with water for injection and the pH value wasadjusted to 5.2.

1.6 L of the Adalimumab 50 mg/mL formulated in water pH 5.2 was refilledin 2 L PETG bottle. The remaining volume of Adalimumab solution wassubjected to DF/UF to increase the concentration to 100 mg/mL.

The Adalimumab 100 mg/mL formulated in water pH 5.3 was sterile filteredand 0.8 L of them was filled in 1 L PETG bottle.

Analytics

-   -   Size exclusion chromatography (SEC)    -   pH—measurement    -   Osmolality measurement    -   Density measurement    -   Protein concentration by means of OD280    -   Optical appearance    -   Ion exchange chromatography (IEC)        Freeze/Thaw Experiment of Adalimumab 1 L Containers

Adalimumab 100° mg/mL formulated in water in 1 L PETG containers wasprecooled to 2-8° C. and than froze at −80° C., freezing cycle >12 hrs.The frozen samples in 1 L PETG bottles were successively thawed at 25°C. in a water bath. During thawing the bottles of the frozen solutionsdipped in the water bath up to liquid level. The following samples werepulled just after thawing without homogenization and afterhomogenization by 15 and 30 turn top over end.

TABLE 68 Sample pull scheme: Turns of each bottle Sample Analyticaltests 1 0 5 mL top protein content, osmolality, 2 0 5 mL middle pH,density, SEC 3 0 5 mL bottom 4 15 5 mL top protein content, osmolality,5 15 5 mL middle pH, density, SEC 6 15 5 mL bottom 7 30 5 mL top proteincontent, osmolality, 8 30 5 mL middle pH, density, SEC and 9 30 5 mLbottom subvisible particlesCharacterization of Adalimumab Solutions

Adalimumab 50 mg/mL and 100° mg/mL formulated in water appeared everytime clearly, light yellow, not opalescent and without wave patternafter gentle movement.

Also after freezing and thawing the Adalimumab formulated in water didnot change the appearance (just after thawing and also after 15 and 30times turn top over end).

A slight wave patterns were seen after gentle movement of the bottlejust after thawing and dipping the needle into the solution duringsample pull just after thawing.

In contrast to similar experiments with Adalimumab in commercial bufferthe Adalimumab solution 50 mg/mL in water did not show any gradient ofprotein concentration, density and osmolality.

The Adalimumab solution 100 mg/mL did also not show any gradient ofprotein concentration, density, osmolality.

Stability was assessed after 6 months storage at −30° C. and −80° C.,respectively.

In the following the respective analytical data are outlined:

TABLE 69 Adalimumab 50 and 100 mg/mL, before freeze/thaw processingprotein content subvisible particles density osmolality (gravimertic) 1mL 1 mL 1 mL pH g/cm3 mOsmol/kg mg/mL >=1 μm >=10 μm >=25 μm 50 mg/mL5.18 1.0121 5 49.3 7953 5 0 in water 100 mg/mL 5.32 1.0262 12 99.8 154 42 in water

TABLE 70 Adalimumab 50 mg/mL, pH 5.2 formulated in water, afterfreeze/thaw processing protein purity subvisible particles densityosmolality content (SEC) 1 mL 1 mL 1 mL turn sample pH g/cm3 mOsmol/kgmg/mL % >=1 μm >=10 μm >=25 μm  0 top 5.20 1.0119 6 48.7 99.597 — — —  0middle 5.19 1.0120 8 49.4 99.576 — — —  0 bottom 5.17 1.0120 6 49.899.649 — — — 15 top 5.20 1.0120 4 49.7 99.649 — — — 15 middle 5.181.0120 5 49.2 99.678 — — — 15 bottom 5.17 1.0120 4 49.1 99.637 — — — 30top 5.19 1.0120 5 49.7 99.647 1280 4 0 30 middle 5.17 1.0120 3 50.499.637 2055 13 0 30 bottom 5.18 1.0120 6 48.9 99.611 3889 37 11

TABLE 71 Adalimumab 100 mg/mL, pH 5.2 formulated in water, afterfreeze/thaw processing protein purity subvisible particles densityosmolality content (SEC) 1 mL 1 mL 1 mL turn sample pH g/cm3 mOsmol/kgmg/mL % >=1 μm >=10 μm >=25 μm  0 top 5.29 1.0259 13 98.7 99.424 — — — 0 middle 5.3 1.0262 16 99.9 99.468 — — —  0 bottom 5.28 1.0262 14 101.299.48 — — — 15 top 5.27 1.0261 13 98.9 99.511 — — — 15 middle 5.271.0261 16 97.7 99.466 — — — — bottom 5.28 1.0261 15 97.0 99.483 — — — 30top 5.29 1.0261 16 96.6 99.439 231 58 49 30 middle 5.28 1.0261 16 97.099.467 169 21 9 30 bottom 5.28 1.0261 16 99.3 99.476 131 3 1

TABLE 72 Adalimumab 100 mg/mL, pH 5.2 formulated in water, stabilityafter storage SEC IEC aggregates sum of monomer lysin visual subvisibleparticles (1 mL) Testing time point fragments isoforms appearance >=1μm >=10 μm >=25 μm T0 0.55 85.523 clear, 155 3 1 99.40 no particular0.05 matter T6 months, −80° C. 0.47 82.124 clear, 210 8 5 99.39 noparticular 0.14 matter T6 months, −30° C. 1.28 81.61 clear, 171 71 5198.58 no particular 0.14 matterConclusion

No significant instabilities of Adalimumab formulated in water at 50 and100 mg/mL after freeze/thaw processing and after storage at −30° C. or−80° C. for up to 6 months have been observed with the analyticalmethodologies applied.

Example 31: Freezing and Thawing Process of Adalimumab in Low-IonicFormulation—Process Design Space Including Protein Content

Preparation of Solution

Adalimumab BDS (Bulk Drug Substance) was thawed in a 23° C. circulatingwater bath. The solution was up-concentrated to a target concentrationof 100 mg/ml for the purpose of volume reduction using aUltrafiltration/Diafiltration (UF/DF) method (Pellicon “Mini” 2). Twocassettes of Millipore Pellicon 2 tangential flow mini-cassettes withBiomax 10K polyethersulfone were installed in the Pellicon 2 unit. Atprocess start the flow rate was measured at 60 ml/min and feed pressurewas 21 psi. The process was stopped at 111.3 mg/ml proteinconcentration.

Spectra/Por molecular porous membrane tubing was used for dialysis(diameter 48 mm, 18 ml/cm volume, 75 cm length). A volume of 8 L ofAdalimumab 100 mg/ml at pH 5.2 were transferred to 8 dialysis tubes.Each tube was filled with 1 L of Adalimumab 100 mg/ml. Four tubes equalto 4 L of solution were placed in a container with 36 L of water forinjection, i.e. a solution exchange factor of 1:10 was accomplished. Thesolution was allowed to reach equilibrium before the volume wasexchanged against fresh water for injection. The solution exchange wasrepeated 5 times until a total solution exchange factor of 1:100,000 wasreached.

After the solution was completely exchanged by dialysis it wasup-concentrated by the second UF/DF step. The second UF/DF step wasperformed like the first step. A final concentration of 247.5 mg/mlAdalimumab in low-ionic formulation was achieved. The UF/DF wasperformed with starting material that already contained polysorbate 80.It could be expected that polysorbate 80 accumulated in the finalprotein solution resulting in a higher polysorbate content than 0.1%.

The up-concentrated bulk solution of 247.5 mg/ml Adalimumab was dilutedwith WFI to lower protein concentration levels as needed—200 mg/ml, 175mg/ml, 150 mg/ml, 140 mg/ml, 130 mg/ml, 120 mg/ml, 100 mg/ml, 80 mg/ml,50 mg/ml, 40 mg/ml, and 25 mg/ml. The bottle fill volume was 1600 ml forall experiments.

Freezing Procedures

A series of increasing freeze rates was used in this evaluation:Ultra-low temperature freezer bottom shelf<Ultra-low temperature freezermiddle shelf<Ultra-low temperature freezer top shelf<<Dry ice.

A <−70° C. freezer was used for the experiments (Capacity: 20.2 Cu. Ft.(572 liters). Three shelves were used. Each was loaded with nine 2 LPETG bottles. The bottles were stored at room temperature before beingplaced in the freezer. Freezing continues for at least 48 hours. For thedesign space evaluations, three positions with increasing freeze rateswere chosen. A front position on the bottom shelf was used for theslowest freeze rate. Faster freeze rates were accomplished at the centerposition on the middle shelf. The fastest freeze rate in the freezersetup was performed in the back/right position on the top shelf.

For freezing by dry ice, one bottle was completely surrounded by dry icefor at least 8 hours. In a Styrofoam box, the bottom was covered with alayer of dry ice (approx. 3 to 5 cm thick). One bottle was placedstanding on top of the dry ice layer. Consequently, the space betweenthe bottle and the inner walls of the styrofoam box was filled with dryice until every surface but the cap was covered. After freezing time,the bottle was removed and thawed immediately or placed in a −70° C.freezer for storage.

Thawing Procedures

A series of thawing rates was used in this evaluation: Cooled air at 4°C.<<Water bath 23° C.<Water bath 37° C.

Analytics

The following analytics were performed to characterize the samples:

-   -   Osmolality    -   Conductivity    -   pH    -   Density    -   Protein concentration by direct UV (280 nm)

For the concentration test, samples were diluted with water until anabsorbance <1.2 was reached. The absorbance coefficient for theAdalimumab molecule at 280 nm of 1.39 was used.

Characterization of Adalimumab Solutions

Bottle mapping studies revealed a slight tendency towards gradientformation in the bottle volume. Especially for the slower freeze andthaw rates, higher protein concentrations were detected near the bottlebottom. This phenomenon was also reflected in conductivity, density, andosmolality data. The pH appears practically constant in all testedconditions.

In previous investigations regarding the Freeze and Thaw design spacefor the bottle based system in ultra-low temperature freezers, theappearance of sedimentation was found to be the main failure modedetermining the boundaries of the allowable operating range. In thisstudy, this boundary was not observed although the investigated designspace covered very wide ranges. The unique behavior of this product isalso reflected in the very low tendency to form concentration gradientsduring this freezing and thawing process. In prior studies it wasconcluded that the product and process inherent gradient formation isthe cause for the appearance of precipitate under certain processconditions. As a result, it was determined that from a processstandpoint this system is feasible for Adalimumab in low-ionicformulation pH 5 up to a bulk drug substance concentration of 247.5mg/ml. The investigated Adalimumab water formulation surprisinglydemonstrated superior performance in comparison to other testedAdalimumab formulations.

TABLE 73 Distribution of Protein Concentration, Conductivity,Osmolarity, Density, and pH in the Freshly Thawed (23° C. water bath)100 mg/ml Adalimumab in Low Ionic Formulation Containing Bottles osmo-conduc- sample volume larity tivity density Adalimumab name ml mosm/kgmS/cm pH g/cm³ conc mg/ml Freeze & Thaw Conditions: −70 C. Top/23 C.Thaw 1  40 11 0.61 5.43 1.021  78.0 2 210 14 0.67 5.43 1.024  92.2 3 22515 0.70 5.43 1.0253  98.1 4 200 17 0.72 5.46 1.0259 103.9 5 175 18 0.735.43 1.0268 100.2 6 180 18 0.74 5.43 1.0275 100.9 7 230 20 0.80 5.461.0264 109.1 8 180 22 0.81 5.45 1.0294 111.2 9 150 21 0.82 5.44 1.0307118.0 Freeze & Thaw Conditions: −70 C. Middle/23 C. Thaw 1  30  8 0.545.43 1.0174  65.3 2 175 18 0.68 5.44 1.0235  91.3 3 200 17 0.70 5.441.0245  92.1 4 185 17 0.72 5.44 1.0249 102.9 5 200 18 0.71 5.43 1.0248 95.6 6 200 20 0.73 5.44 1.0262  96.6 7 175 20 0.74 5.44 1.0283 107.5 8180 20 0.77 5.45 1.0306 116.1 9 200 26 0.82 5.44 1.0346 131.1 Freeze &Thaw Conditions: −70 C. Bottom/23 C. Thaw 1  35  9 0.60 5.41 1.0195 73.2 2 200 13 0.68 5.41 1.0231  89.6 3 225 16 0.70 5.41 1.0241  93.2 4180 15 0.71 5.41 1.0246  96.8 5 200 15 0.72 5.40 1.0249  95.7 6 200 190.73 5.41 1.0259  96.4 7 185 21 0.75 5.42 1.0272 102.6 8 200 26 0.795.41 1.0309 116.8 9 175 31 0.85 5.42 1.0372 141.5

TABLE 74 Distribution of Protein Concentration, Conductivity,Osmolarity, Density, and pH in the Freshly Thawed (23° C. water bath)140 mg/ml Adalimumab in Low Ionic Formulation Containing Bottles osmo-conduc- sample volume larity tivity density Adalimumab name ml mosm/kgmS/cm pH g/cm³ conc mg/ml Freeze & Thaw Conditions: −70 C. Top/23 C.Thaw 1  50 36 0.87 5.43 1.0338 130.0 2 215 42 0.90 5.43 1.0354 139.9 3170 54 0.91 5.43 1.0362 144.9 4 210 41 0.84 5.44 1.0365 141.5 5 200 400.93 5.43 1.0364 157.7 6 200 41 0.92 5.43 1.0364 140.0 7 190 41 0.925.43 1.0363 143.4 8 180 44 0.82 5.43 1.037 150.1 9 140 45 0.95 5.411.038 148.3 Freeze & Thaw Conditions: −70 C. Middle/23 C. Thaw 1  25 320.81 5.45 1.0284 112.0 2 175 34 0.84 5.44 1.0307 122.4 3 175 36 0.885.44 1.033 133.9 4 200 40 0.90 5.43 1.0342 134.6 5 220 40 0.92 5.431.0351 140.9 6 185 45 0.94 5.43 1.0369 143.6 7 210 47 0.97 5.43 1.0384149.8 8 175 47 0.99 5.43 1.0399 160.3 9 190 48 1.01 5.43 1.0435 168.3Freeze & Thaw Condition: −70 C. bottom/23 C. Thaw 1  75 28 0.75 5.451.0257  88.7 2 180 34 0.82 5.46 1.029 111.6 3 175 34 0.84 5.44 1.0313123.5 4 220 37 0.86 5.44 1.0322 118.8 5 165 38 0.89 5.45 1.0337 126.3 6215 44 0.95 5.45 1.0374 137.6 7 210 49 1.00 5.45 1.0407 149.6 8 150 531.03 5.43 1.0429 154.9 9 180 60 1.06 5.44 1.0501 183.2

TABLE 75 Distribution of Protein Concentration, Conductivity,Osmolarity, Density, and pH in the Freshly Thawed (37° C. water bath)200 mg/ml Adalimumab in Low Ionic Formulation Containing Bottles osmo-conduc- sample volume larity tivity density Adalimumab name ml mosm/kgmS/cm pH g/cm³ conc mg/ml Freeze & Thaw Conditions: −70 C. Top/37 C.Thaw 1  40 37 0 89 5.26 1.0573 197.5 2 210 35 0.96 5.24 1.0573 195.3 3175 34 0.96 5.22 1.0578 200.3 4 210 36 0.88 5.27 0.0579 193.9 5 210 390.89 5.24 0.058 210.6 6 190 39 0.84 5.27 0.058 213.8 7 200 41 0 88 5.270.058 206.7 8 170 41 0.88 5.24 0.0581 196.6 9 160 39 0.89 5.29 0.0595201.9 Freeze & Thaw Conditions: −70 C. Center/37 C. Thaw 1  10 31 0.855.29 1.0485 170.2 2 185 35 0.89 5.31 1.0505 189.3 3 215 37 0.90 5.331.0518 191.7 4 185 38 0.89 5.27 1.0519 191.8 5 200 36 0.90 5.32 1.0528196.3 6 200 48 0.90 5.23 1.0533 189.3 7 170 37 0.90 5.23 1.0536 193.1 8215 39 0.91 5.33 1.0552 202.1 9 180 48 0.92 5.31 1.0613 225.5 Freeze &Thaw Conditions: −70 C. Bottom/37 C. Thaw 1  50 22 0.96 5.27 1.0361107.1 2 185 29 0.83 5.26 1.0422 163.0 3 180 38 0.88 5.27 1.0522 201.2 4185 41 0.90 5.24 1.0535 198.9 5 180 44 0.92 5.28 1.0552 201.4 6 195 400.91 5.32 1.0558 201.7 7 180 40 0.91 5.32 1.0560 199.6 8 175 41 0.855.26 1.0568 206.2 9 190 48 0.91 5.3 1.0619 229.3

TABLE 76 Distribution of Protein Concentration, Conductivity,Osmolarity, Density, and pH in the Freshly Thawed (23° C. water bath)247.5 mg/ml Adalimumab in Low Ionic Formulation Containing Bottles osmo-conduc- sample volume larity tivity density Adalimumab name ml mosm/kgmS/cm pH g/cm³ conc mg/ml Freeze & Thaw Conditions: −70 C. Top/ 23 C.Thaw 1  55 46 0.98 5.28 1.0755 260.9 2 190 72 0.97 5.28 1.0751 270 8 3190 56 0.97 5.28 1 0751 314.7 4 200 49 0.96 5.27 1.0751 274.8 5 200 580.96 5.27 1.0752 278.4 6 210 57 0.97 5.28 1.0752 275.0 7 210 76 0.965.28 1.0748 276.5 8 175 75 0.96 5.27 1.0754 274.5 9 150 62 0.97 5.281.0763 276.3 Freeze & Thaw Conditions: −70 C. Middle/23 C. Thaw 1  30 370.95 5.29 1.0671 250.0 2 200 59 0.95 5.32 1.0704 251.3 3 175 51 0.975.31 1.0722 262.7 4 215 56 0.93 5.31 1.073 327.1 5 200 48 0.99 5.311.0739 267.1 6 200 67 0.98 5.31 1.0744 270.6 7 230 59 0.95 5.32 1.0753273.2 8 175 70 0.96 5.32 1.0771 273.3 9 175 83 0.96 5.32 1.0825 289.6Freeze & Thaw Conditions: −70 C. Bottom/23 C. Thaw 1  50 32 0.92 5.241.0632 215.3 2 220 59 0.95 5.27 1.069 221.7 3 175 72 0.96 5.27 1.0708268.1 4 180 58 0.96 5.27 1.0725 260.7 5 210 63 0.96 5.27 1.0729 266.8 6150 69 0.96 5.28 1.0744 280.3 7 225 50 0.96 5.29 1.0762 280.3 8 200 680.95 5.28 1.0789 288.6 9 180 70 0.95 5.29 1.0846 293.0

TABLE 77 Distribution of Protein Concentration, Conductivity,Osmolarity, Density, and pH in the Freshly Thawed (23° C. water bath)247.5 mg/ml Adalimumab in Low Ionic Formulation Containing Bottles AfterDry Ice Freezing osmo- conduc- sample volume larity tivity densityAdalimumab name ml mosm/kg mS/cm pH g/cm³ conc mg/ml Freeze & ThawConditions: Dry Ice Freeze/23 C. Thaw 1  50 51 0.94 5.28 1.0643 258.9 2210 68 0.94 5.29 1.0683 261.9 3 180 50 0.95 5.29 1.0702 251.7 4 190 690.95 5.29 1.0732 262.2 5 210 72 0.96 5.31 1.0738 274.4 6 225 63 0.95 5.31.0746 265.7 7 160 57 0.95 5.3 1.0747 261.9 8 190 63 0.95 5.31 1.0749270.9 9 200 50 0.95 5.31 1.075 271.4 Freeze & Thaw Conditions: Dry IceFreeze/2-8 C. Thaw 1  50 44 0.96 5.29 1.0665 263.1 2 190 53 0.96 5.311.0684 258.1 3 200 56 0.96 5.30 1.0691 247.6 4 200 58 0.96 5.30 1.0693262.2 5 190 64 0.95 5.31 1.0695 243.2 6 200 61 0.95 5.3 1.0695 266.8 7175 49 0.96 5.32 1.0697 256.2 8 200 50 0.95 5.31 1.0697 261.2 9 175 480.96 5.32 1.0704 247.1

APPENDIX A PCS DATA concentration z- peak con- [mg/ml] z- average peakmonomer centration averagne average average monomer average [mg/ml]value [nm] value [nm] [nm] value [nm] Adalimumab 9.35 9.35 2.08 2.082.55 2.55 23.40 23.27 2.30 2.47 2.81 2.87 22.70 2.77 3.01 23.70 2.362.78 34.80 34.20 1.56 1.55 1.85 1.87 35.70 1.54 1.82 32.10 1.56 1.9335.40 36.10 1.61 1.63 1.92 1.92 36.10 1.64 1.93 36.80 1.63 1.92 42.1043.00 1.75 1.75 2.12 2.12 45.60 1.78 2.15 41.30 1.74 2.10 60.20 57.402.06 2.02 2.27 2.37 55.90 2.04 2.45 56.10 1.98 2.39 63.20 65.87 2.112.24 2.52 2.67 71.70 2.49 2.89 62.70 2.13 2.61 73.40 75.13 2.38 2.412.83 2.89 75.60 2.51 3.01 76.40 2.35 2.82 78.60 78.07 2.53 2.55 2.992.99 78.80 2.62 3.01 76.80 2.50 2.96 90.40 95.73 2.80 2.85 3.35 3.41107.40 2.99 3.55 89.40 2.76 3.33 96.20 94.77 2.88 2.86 3.50 3.50 96.002.91 3.61 92.10 2.80 3.38 201.00 206.63 4.52 4.82 5.22 5.74 227.50 5.046.12 191.40 4.89 5.89 J695 9.99 9.99 2.28 2.28 1.66 1.66 19.31 19.292.05 2.12 1.81 1.81 19.26 2.30 1.79 19.29 2.02 1.84 29.59 29.40 1.781.62 1.10 1.16 29.7 1.51 1.15 28.91 1.56 1.22 37.97 37.55 1.51 1.56 1.221.23 38.02 1.67 1.22 36.65 1.49 1.24 49.15 46.32 1.64 1.58 1.31 1.2945.95 1.57 1.30 43.87 1.53 1.26 58.75 56.18 1.60 1.61 1.49 1.47 55.021.71 1.38 54.76 1.53 1.53 77.69 77.81 2.64 2.43 2.73 2.61 77.62 2.312.57 78.13 2.35 2.52 94.45 97.65 2.11 2.07 2.05 2.05 105.06 2.14 2.0593.45 1.97 2.04 116.37 114.52 3.69 2.69 1.95 2.00 113.92 2.25 2.06113.27 2.13 1.99 121.21 133.25 9.78 9.49 11.50 11.00 139.8 9.63 11.10138.73 9.06 10.40 226.67 217.53 4.94 5.34 4.72 4.84 216.1 6.01 5.25209.83 5.06 4.55 Human Serum Albumin 9.88 9.88 14.90 14.90 2.32 2.3222.94 22.89 8.26 8.29 1.2 1.18 22.73 8.28 1.18 23.00 8.33 1.17 36.7836.47 7.40 7.44 1.22 1.23 37.33 7.80 1.24 35.29 7.12 1.22 45.97 46.067.09 6.92 1.27 1.25 47.61 6.54 1.24 44.61 7.13 1.25 58.47 58.56 5.946.13 1.3 1.31 62.69 6.04 1.31 54.52 6.41 1.32 61.89 60.31 5.83 6.14 1.331.32 59.76 6.57 1.34 59.28 6.01 1.29 75.37 76.24 5.58 5.46 1.4 1.4083.69 5.14 1.45 69.67 5.67 1.36 92.90 85.87 5.30 5.14 1.49 1.47 84.225.05 1.49 80.50 5.08 1.43 115.93 112.74 4.78 4.94 1.68 1.61 110.00 5.041.58 112.30 4.99 1.57 182.79 177.69 9.85 9.13 2.27 2.19 178.24 9.29 2.21172.05 8.26 2.08

APPENDIX B SEC DATA mean mean mean mean conc. conc. monomer monomeraggregate aggregate fragment fragment [mg/ml] [mg/ml] [%] [%] [%] [%][%] [%] Adalimumab 9.35 9.35 99.40 99.40 0.50 0.50 0.10 0.10 23.40 23.2799.60 99.57 0.40 0.40 0.10 0.10 22.70 99.50 0.40 0.10 23.70 99.60 0.400.10 34.80 34.20 99.50 99.47 0.50 0.47 0.10 0.10 35.70 99.40 0.50 0.1032.10 99.50 0.40 0.10 35.40 36.10 99.40 99.40 0.60 0.53 0.10 0.10 36.1099.40 0.50 0.10 36.80 99.40 0.50 0.10 42.10 43.00 99.40 99.33 0.50 0.570.10 0.10 45.60 99.30 0.60 0.10 41.30 99.30 0.60 0.10 60.20 57.40 99.3099.30 0.60 0.60 0.10 0.10 55.90 99.30 0.60 0.10 56.10 99.30 0.60 0.1063.20 65.87 99.30 99.27 0.60 0.67 0.10 0.10 71.70 99.20 0.70 0.10 62.7099.30 0.70 0.10 73.40 75.13 99.20 99.23 0.70 0.70 0.10 0.10 75.60 99.200.70 0.10 76.40 99.30 0.70 0.10 78.60 78.07 99.30 99.30 0.60 0.60 0.100.10 78.80 99.30 0.60 0.10 76.80 99.30 0.60 0.10 90.40 95.73 99.20 99.130.80 0.80 0.10 0.10 107.40 99.10 0.80 0.10 89.40 99.10 0.80 0.10 96.2094.77 99.10 99.03 0.80 0.87 0.10 0.10 96.00 99.00 0.90 0.10 92.10 99.000.90 0.10 201.00 206.63 98.80 98.80 1.10 1.10 0.10 0.10 227.50 98.801.10 0.10 191.40 98.80 1.10 0.10 J695 9.99 9.99 99.39 99.39 0.44 0.440.17 0.17 19.31 19.29 99.38 99.38 0.44 0.44 0.18 0.19 19.26 99.37 0.440.20 19.29 99.38 0.44 0.18 29.59 29.40 99.31 99.33 0.51 0.50 0.18 0.1829.70 99.32 0.50 0.18 28.91 99.35 0.48 0.17 37.97 37.55 99.31 99.29 0.520.52 0.17 0.19 38.02 99.27 0.52 0.21 36.65 99.30 0.51 0.19 49.15 46.3299.19 99.20 0.60 0.60 0.21 0.20 45.95 99.20 0.60 0.20 43.87 99.20 0.610.19 58.75 56.18 99.16 99.16 0.64 0.64 0.21 0.21 55.02 99.17 0.64 0.2054.76 99.15 0.63 0.22 77.69 77.81 99.11 99.10 0.70 0.70 0.19 0.20 77.6299.09 0.69 0.22 78.13 99.10 0.70 0.20 94.45 97.65 99.05 99.06 0.72 0.710.23 0.22 105.06 99.06 0.72 0.21 93.45 99.07 0.70 0.23 116.37 114.5298.94 98.91 0.85 0.88 0.21 0.22 113.92 98.91 0.88 0.22 113.27 98.89 0.900.22 121.21 133.25 98.87 98.89 0.91 0.90 0.22 0.22 139.80 98.89 0.890.22 138.73 98.90 0.89 0.21 226.67 217.53 98.58 98.57 1.19 1.21 0.240.23 216.10 98.58 1.18 0.24 209.83 98.54 1.25 0.21 Human Serum AlbuminPeak 1 Peak 2 Peak 3 Peak 4 (HSA) Area Area Area Area Area Area AreaArea sample [mVs] [%] [mVs] [%] [mVs] [%] [mVs] [%] sample 1 59.7102.312 2.975 0.115 43.159 1.671 2477.282 95.902 C = 9.88 mg/ml sample 2102.785 2.685 7.859 0.205 73.588 1.923 3643.350 95187 C = 22.94 mg/mlsample 3 124.226 3.071 11.038 0.273 83.310 2.059 3826.908 94.597 C =22.73 mg/ml sample 4 138.353 3.266 14.525 0.343 88.429 2.087 3994.99094.304 C = 23.00 mg/ml sample 5 147.465 3.459 14.537 0.341 91.304 2.1414010.385 94.059 C = 36.78 mg/ml sample 6 153.956 3.552 14.707 0.33994.093 2.171 4071.680 93.938 C = 37.33 mg/ml sample 7 171.478 3.60816.064 0.338 105.244 2.214 4459.830 93.839 C = 35.29 mg/ml sample 8180.027 3.675 17.392 0.355 109.717 2.239 4592.102 93.731 C = 45.97 mg/mlsample 9 193.325 3.719 19.206 0.370 116.474 2.241 4868.705 93.670 C =47.61 mg/ml sample 10 191.512 3.799 19.167 0.380 112.261 2.227 4718.55493.594 C = 44.61 mg/ml sample 11 215.044 4.026 17.870 0.335 118.4812.218 4989.978 93.421 C = 58.47 mg/ml sample 12 218.072 4.037 20.0880.372 122.251 2.263 5041.542 93.328 C = 62.69 mg/ml sample 13 228.0144.053 19.957 0.355 126.583 2.250 5251.513 93.343 C = 54.52 mg/ml sample14 231.235 4.085 22.518 0.398 127.330 2.250 5279.038 93.267 C = 61.89mg/ml sample 15 237.894 4100 22.939 0.395 130.352 2.246 5411.384 93.258C = 59.76 mg/ml sample 16 202.103 4.139 17.178 0.352 108.780 2.2284554.912 93.282 C = 59.28 mg/ml sample 17 230.552 4.196 18.565 0.338123.207 2.242 5122.467 93.224 C = 75.37 mg/ml sample 18 215.365 4.16218.136 0.351 110.152 2.129 4830.372 93.358 C = 83.69 mg/ml sample 21233.866 4.316 21.951 0.405 116.325 2.147 5046.183 93.132 C =84.22 mg/mlsample 22 221.816 4.461 18.940 0.381 111.006 2.232 4620.655 92.926 C =80.50 mg/ml sample 23 223.187 4.783 16.684 0.358 104116 2.231 4322.73292.629 C = 115.93 mg/ml sample 24 209.281 4.718 18.745 0.423 96.430 21744111.363 92.686 C = 110.00 mg/ml sample 25 172.657 4.537 15.457 0.40680.850 2.125 3536.192 92.932 C = 112.30 mg/ml sample 26 178.208 4.95015.254 0.424 80.906 2.247 3325.648 92.379 C = 182.79 mg/ml sample 27194.516 4.814 17.323 0.429 90.433 2.238 3738.717 95.520 C = 178.24 mg/mlsample 28 79.605 2.103 12.876 0.340 74.965 1.981 3617.238 95.576 C =172.05 mg/ml

APPENDIX C IEC DATA Adalimumab mean sum mean conc. conc. Lysin sum[mg/ml] [mg/ml] [%] [%] 9.35 9.35 86.09 86.09 23.40 23.27 86.15 86.1322.70 86.12 23.70 86.13 34.80 34.20 86.15 86.11 35.70 86.11 32.10 86.0635.40 36.10 86.03 86.04 36.10 86.06 36.80 86.03 42.10 43.00 85.98 85.9645.60 85.95 41.30 85.95 60.20 57.40 85.97 85.96 55.90 85.94 56.10 85.9763.20 65.87 85.96 85.94 71.70 85.97 62.70 85.90 73.40 75.13 85.99 85.9775.60 85.98 76.40 85.95 78.60 78.07 86.00 85.97 78.80 85.97 76.80 85.9490.40 95.73 85.96 85.92 107.40 85.97 89.40 85.83 96.20 94.77 85.93 85.8896.00 85.87 92.10 85.84 201.00 206.63 85.88 85.90 227.50 85.97 191.4085.84 J695 sum mean sum sum mean sum sum mean sum mean peak peak acidicacidic basic basic conc. conc. 1-7 1-7 peaks peaks peaks peaks [mg/ml][mg/ml] [%] [%] [%] [%] [%] [%] 9.99 9.99 89.24 89.24 10.24 10.24 0.520.52 19.31 19.29 89.32 89.28 10.19 10.21 0.50 0.51 19.26 89.23 10.260.52 19.29 89.30 10.19 0.51 29.59 29.40 89.33 89.30 10.14 10.17 0.540.53 29.70 89.26 10.20 0.54 28.91 89.32 10.16 0.52 37.97 37.55 89.3289.30 10.13 10.15 0.56 0.55 38.02 89.27 10.18 0.55 36.65 89.31 10.150.55 49.15 46.32 89.07 89.10 10.40 10.37 0.53 0.53 45.95 89.12 10.340.54 43.87 89.12 10.36 0.53 58.75 56.18 89.13 89.17 10.36 10.31 0.520.53 55.02 89.21 10.27 0.52 54.76 89.18 10.29 0.54 77.69 77.81 89.2289.17 10.25 10.29 0.53 0.54 77.62 89.09 10.36 0.55 78.13 89.20 10.260.55 94.45 97.65 89.20 89.16 10.28 10.30 0.52 0.54 105.06 89.12 10.330.55 93.45 89.16 10.29 0.55 116.37 114.52 89.03 89.08 10.41 10.36 0.560.55 113.92 89.15 10.31 0.54 113.27 89.06 10.37 0.56 121.21 133.25 89.2689.13 10.20 10.33 0.54 0.55 139.80 89.07 10.38 0.56 138.73 89.05 10.400.55 226.67 217.53 88.72 88.78 10.69 10.63 0.59 0.59 216.10 88.82 10.600.58 209.83 88.81 10.60 0.59

APPENDIX D Duration of 63 mg/ml 220 mg/ml Test Item Component Testing 5°C. 5° C. Clarity and Turbidity Initial 3.6 8.0 opalescence 1 month 3.58.0 3 month 3.5 7.4 Degree of B scale Initial <B9 =B9 coloration of 1month <B9 <B8 liquids 3 month <B9 <B7 pH Single value Initial 5.3 5.4 1month 5.3 5.4 3 month 5.3 5.4 Particulate visual score Initial 2.2 0.2contamination: 1 month 2.2 0.4 visible particles 3 month 2.1 0.2Particulate Particles >= 10 Initial 181 357 contamination: μm[/Container] 1 month 423 290 subvisible 3 month 216 1762 particlesParticles >= 25 Initial 15 3 μm [/Container] 1 month 11 18 3 month 2 50Size exclusion Principal peak Initial 0.2 0.5 chromatography (aggregate)[%] 1 month 0.2 0.6 (SE-HPIC) 3 month 0.2 0.7 Principal peak Initial99.8 99.4 (monomer) [%] 1 month 99.7 99.3 3 month 99.7 99.2 Principalpeak Initial 0.1 0.1 (fragment)[%] 1 month 0.1 0.1 3 month 0.0 0.0Cation exchange 1st acidic region Initial 2.2 2.2 HPIC (CEX- [%] 1 month2.2 2.2 HPIC) 3 month 2.1 2.0 2nd acidic Initial 10.4 10.3 region[%] 1month 10.2 10.0 3 month 10.4 10.2 Sum of lysine Initial 86.0 86.1variants [%] 1 month 85.9 85.9 3 month 86.2 86.1 Peak between Initial0.8 0.8 lysine 1 und 1 month 1.0 1.0 lysine 2 [%] 3 month 0.8 0.8 Peaksafter Initial 0.5 0.6 Lysin 2 [%] 1 month 0.7 0.9 3 month 0.5 0.8Duration 63 mg/ml 220 mg/ml of 25° C./60% 25° C./60% Test Item ComponentTesting R.H. R.H. Clarity and Turbidity Initial — — opalescence 1 month3.51 8.55 3 month 3.70 7.56 Degree of B scale Initial — — coloration of1 month <B9 <B8 liquids 3 month <B9 <B7 pH Single value Initial — — 1month 5.4 5.4 3 month 5.3 5.4 Particulate visual score Initial — —contamination: 1 month 2.5 0.7 visible particles 3 month 3.4 0.0Particulate Particles >= 10 Initial — — contamination: μm [/Container] 1month 412 490 subvisible 3 month 277 4516 particles Particles >= 25Initial — — μm [/Container] 1 month 10 14 3 month 7 128 Size exclusionPrincipal peak Initial — — chromatography (aggregate) [%] 1 month 0.30.8 (SE-HPIC) 3 month 0.4 1.1 Principal peak Initial — — (monomer) [%] 1month 99.6 99.0 3 month 99.4 98.6 Principal peak Initial — — (fragment)[%] 1 month 0.2 0.2 3 month 0.2 0.2 Cation exchange 1st acidic regionInitial — — HPIC (CEX- [%] 1 month 2.5 2.4 HPIC) 3 month 3.4 3.2 2ndacidic Initial — — region[%] 1 month 11.7 11.4 3 month 15.3 14.9 Sum oflysine Initial — — variants [%] 1 month 83.6 83.8 3 month 79.2 79.2 Peakbetween Initial — — lysine 1 und 1 month 1.2 1.3 lysine 2 [%] 3 month1.3 1.3 Peaks after Initial — — Lysin 2 [%] 1 month 0.9 1.1 3 month 0.81.4 Duration 63 mg/ml 220 mg/ml of 40° C./75% 40° C./75% Test ItemComponent Testing R.N. R.N. Clarity and Turbidity Initial — —opalescence 1 month 3.93 7.80 3 month 3.70 8.10 Degree of B scaleInitial — — coloration of 1 month =B9 =B8 liquids 3 month <B8 <B7 pHSingle value Initial — — 1 month 5.3 5.4 3 month 5.3 5.4 Particulatevisual score Initial — — contamination: 1 month 6.7 0.5 visibleparticles 3 month 17.5 0.4 Particulate Particles >= 10 Initial — —contamination: μm [/Container] 1 month 1088 518 subvisible 3 month 166612 particles Particles >= 25 Initial — — μm [/Container] 1 month 16 143 month 11 30 Size exclusion Principal peak Initial — — chromatography(aggregate) [%] 1 month 0.4 1.4 (SE-HPIC) 3 month 0.8 2.5 Principal peakInitial — — (monomer) [%] 1 month 99.0 98.0 3 month 97.8 96.0 Principalpeak Initial — — (fragment)[%] 1 month 0.6 0.6 3 month 1.4 1.5 Cationexchange 1st acidic region Initial — — HPIC (CEX- [%] 1 month 6.7 6.8HPIC) 3 month 17.5 17.4 2nd acidic Initial — — region[%] 1 month 25.123.6 3 month 40.9 38.6 Sum of lysine Initial — variants [%] 1 month 64.562.0 3 month 36.0 36.0 Peak between Initial — — lysine 1 und 1 month 2.22.5 lysine 2 [%] 3 month 2.9 3.1 Peaks after Initial — — Lysin 2 [%] 1month 1.5 5.2 3 month 1.7 4.8

APPENDIX E Dura- tion LI LI LI LI LI LI LI LI LI LI LI LI LI LI LI LITest Compo- of 50 50 50 50 50 50 50 50 200 200 200 200 200 200 200 200Item nent testing 01*² 02*² 03*² 04*² 05*² 06*² 07*² 08*² 01*² 02*² 03*²04*² 05*² 06*² 07*² 08*² 2-8° C. Clarity Absorp- initial 0.096 0.0960.095 0.100 0.104 0.105 0.099 0.107 0.181 0.187 0.182 0.192 0.184 0.197 0.191  0.199 and tion 1 0.102 0.102 0.093 0.094 0.099 0.101 0.097 0.1000.181 0.185 0.189 0.181 0.190 0.180  0.192  0.192 opales- (340 nm) monthcence Degree visual initial clear and colorless of color- 1 clear andcolorless ation month pH Single initial 5.4 5.4 5.4 5.4 5.4 5.4 5.4 5.45.6 5.6 5.6 5.6 5.6 5.5,  5.6  5.5, value 1 5.4 5.5, 5.5, 5.4 5.5, 5.45.5, 5.5, 5.6 5.6 5.5, 5.5, 5.6 5.5,  5.6  5.6 month Size Principalinitial 0.1 0.1 0.1 0.1 0.1 0.1 0.2 0.2 0.5 0.5 0.5 0.5 0.5 0.5  0.5 0.6 exclu- peak 1 0.1 0.1 0.1 0.1 0.1 0.1 1.7 0.2 0.6 0.6 0.6 0.6 0.60.6  0.7  0.7 sion (aggregate) month chroma- [%] tography Principalinitial 99.6 99.6 99.6 99.6 99.6 99.6 99.7 99.7 99.3 99.3 99.3 99.3 99.399.3 99.3 99.3 (SE- peak HPLC (monomer) [%] 1 99.7 99.7 99.7 99.7 99.799.7 99.7 99.7 99.3 99.3 99.2 99.3 99.3 99.2 99.2 99.2 month Principalinitial 0.3 0.3 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2  0.2 0.1 peak 1 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2  0.2 0.2 (fragment) month [%] Cation 1^(st) acidic initial 3.8 3.7 3.7 3.73.7 3.9 3.8 3.8 3.7 3.8 3.6 4.5 3.9 2.7  2.7  2.8 ex- region [%] 1 3.43.4 3.4 3.4 3.4 3.5 3.4 3.4 3.4 3.4 3.5 3.5 3.5 3.5  3.5  3.5 changemonth HPLC 2^(nd) cidic initial 10.9 10.7 10.4 10.5 10.4 101 10.3 10.210.4 10.2 98 10.1 95 11.6 11.5 11.3 (CEX- region [%] 1 9.2 9.2 9.2 9.29.2 9.2 9.2 9.3 9.1 9.0 9.1 9.1 9.0 9.1  9.1  9.1 HPLC) month Sum ofinitial 83.8 84.2 84.4 84.3 84.4 84.6 84.4 84.6 84.4 84.6 85.2 83.9 85.284.4 84.3 84.5 lysine 1 86.0 86.0 86.0 86.0 86.0 85.9 86.1 86.0 86.386.1 86.1 86.2 86.0 86.0 86.1 86.1 variants month [%] Peak initial 0.98.3 0.8 0.9 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.8  0.8  0.8 between 10.9 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.6 0.8 0.6 0.6 0.8 0.7  0.6  0.6 lysine1 month and lysine 2 [%] Peaks initial 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.60.6 0.6 0.6 0.7 0.6 0.6  0.6  0.6 after 1 0.6 0.5 0.5 0.5 0.5 0.5 0.50.5 0.7 0.7 0.7 0.7 0.7 0.7  0.8  0.7 Lysin 2 month [%] 25° C. ClarityAbsorp- initial 0.096 0.096 0.095 0.100 0.104 0.105 0.099 0.107 0.1810.187 0.182 0.192 0.184 0.197  0.191  0.199 and tion 1 0.106 0.109 0.0960.099 0.104 0.104 0.096 0.105 0.178 0.177 0.198 0.189 0.200 0.194  0.194 0.172 opales- (340 nm) month cence Degree visual initial clear andcolorless of color- 1 clear and colorless ation month pH Single initial5.4 5.4 5.4 5.4 5.4 5.4 5.4 5.4 5.6 5.6 5.6 5.6 5.6 5.5  5.6  5.5 value1 5.4 5.4 5.4 5.4 5.4 5.4 5.4 5.4 5.5 5.6 5.6 5.5 5.5 5.5  5.5  5.5month Size Principal initial 0.1 0.1 0.1 0.1 0.1 0.1 0.2 0.2 0.5 0.5 0.50.5 0.5 0.5  0.5  0.6 exclu- peak 1 0.2 0.2 0.2 0.2 0.2 ? 0.2 0.2 0.80.8 0.8 0.8 0.8 0.8  0.9  0.9 sion (aggregate) month chroma- [%]tography Principal initial 99.6 99.6 99.6 99.6 99.6 99.6 99.7 99.7 99.399.3 99.3 99.3 99.3 99.3 99.3 99.3 (SE- peak 1 99.6 99.6 99.6 99.6 99.6? 99.5 99.5 99.0 99.0 99.0 99.0 99.0 99.0 98.9 98.9 HPLC (monomer) month[%] Principal initial 0.3 0.3 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.20.2 0.2  0.2  0.1 peak 1 0.2 0.2 0.2 0.2 0.2 ? 0.2 0.2 0.2 0.2 0.2 0.20.2 0.2  0.2  0.2 (fragment) month [%] Cation 1^(st) acidic initial 3.83.7 3.7 3.7 3.7 3.9 3.8 3.8 3.7 3.8 3.6 4.5 3.9 2.7  2.7  2.8 ex- region[%] 1 4.1 4.2 4.1 4.1 4.2 4.2 4.1 4.2 4.0 4.0 4.1 4.1 4.0 4.1  4.1  4.0change month HPLC 2^(nd) cidic initial 10.9 10.7 10.4 10.5 10.4 10.110.3 10.2 10.4 10.2 9.8 10.1 9.5 11.6 11.5 11.3 (CEX- region [%] 1 10.910.9 110 10.9 11.0 11.0 11.0 11.1 10.6 10.7 10.7 10.7 10.7 10.7 10.710.9 HPLC) month Sum of initial 83.8 84.2 84.4 84.3 84.4 84.6 84.4 84.684.4 84.6 85.2 83.9 85.2 84.4 84.3 84.5 lysine 1 83.3 83.4 83.3 83.383.2 83.2 83.3 83.1 83.4 83.6 83.4 83.6 83.6 83.5 83.3 83.4 variantsmonth [%] Peak initial 0.9 83 0.8 0.9 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.80.8 0.8  0.8  0.8 between 1 1.0 0.9 1.0 1.0 1.0 0.9 0.9 1.0 1.0 0.8 0.80.7 0.7 0.8  0.9  0.8 lysine 1 month and lysine 2 [%] Peaks initial 0.60.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.7 0.6 0.6  0.6  0.6 after 10.7 0.6 0.7 0.7 0.7 0.6 0.6 0.6 0.9 0.9 0.9 0.9 0.9 0.9  0.9  0.9 Lysin2 month [%] 40° C. Clarity Absorp- initial 0.096 0.096 0.095 0.100 0.1040.105 0.099 0.107 0.181 0.187 0.182 0.192 0.184 0.197  0.191  0.199 andtion 1 0.100 0.110 0.099 0.101 0.111 0.116 0.105 0.113 0.204 0.202 0.1980.205 0.199 0.216  0.209  0.224 opales- (340 nm) month cence Degreevisual initial clear and colorless of color- 1 clear and colorless ationmonth pH Single initial 5.40 5.41 5.41 5.40 5.40 5.40 5.41 5.41 5.575.56 5.55 5.56 5.55 5.54  5.55  5.52 value 1 5.42 5.42 5.43 5.43 5.445.43 5.44 5.44 5.54 5.55 5.55 5.55 5.55 5.55  5.53  5.54 month SizePrincipal initial 0.106 0.104 0.119 0.136 0.139 0.145 0.158 0.159 0.4890.509 0.491 0.484 0.492 0.488  0.515  0.575 exclu- peak 1 0.324 0.3360.322 0.334 0.347 0.349 0.411 0.444 1.308 1.343 1.331 1.340 1.341 1.374 1.453  1.521 sion (aggregate) month chroma- [%] tography Principalinitial 99.605 99.629 99.632 99.626 99.619 99.626 99.653 99.655 99.29499.296 99.348 99.333 99.313 99.349 99.320 99.290 (SE- peak 1 98.92498.914 98.931 98.914 98.895 98.894 98.845 98.782 97.920 97.876 97.89297.901 97.898 97.861 97.752 97.701 HPLC (monomer) month [%] Principalinitial 0.289 0.267 0.249 0.238 0.242 0.229 0.189 0.186 0.218 0.1960.161 0.183 0.195 0.163  0.165  0.135 peak 1 0.752 0.751 0.747 0.7520.758 0.757 0.744 0.773 0.773 0.781 0.777 0.759 0.762 0.765  0.794 0.779 (fragment) month [%] Cation 1^(st) acidic initial 3.8 3.7 3.7 3.73.7 3.9 3.8 3.8 3.7 3.8 3.6 4.5 3.9 2.7  2.7  2.8 ex- region [%] 1 5.45.8 5.3 5.8 5.4 5.8 5.4 5.5 5.3 5.6 5.3 5.6 5.3 5.6  5.3  5.4 changemonth HPLC 2^(nd) cidic initial 10.9 10.7 10.4 10.5 10.4 10.1 10.3 10.210.4 10.2 9.8 10.1 9.5 11.6 11.5 11.3 (CEX- region [%] 1 29.8 29.8 29.729.7 29.8 29.8 30.2 30.7 28.6 28.9 28.5 28.7 28.6 28.9 29.1 29.2 HPLC)month Sum of initial 83.8 84.2 84.4 84.3 84.4 84.6 84.4 84.6 84.4 84.685.2 83.9 85.2 84.4 84.3 84.5 lysine 1 61.2 61.0 61.3 61.0 61.2 60.960.9 60.5 62.0 61.7 62.0 61.8 62.0 61.6 61.5 61.4 variants month [%]Peak initial 0.9 8.3 0.8 0.9 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.8  0.8 between 1 2.3 2.1 2.3 2.2 2.2 2.2 2.1 2.0 2.2 2.0 2.2 2.0 2.22.0  2.0  2.0 lysine 1 month and lysine 2 [%] Peaks initial 0.6 0.6 0.60.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.7 0.6 0.6  0.6  0.6 after 1 1.3 1.31.3 1.3 1.4 1.3 1.4 1.4 1.9 1.9 2.0 1.9 1.9 1.9  2.0  2.0 Lysin 2 month[%]

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims. The contents of allreferences, patents and published patent applications cited throughoutthis application are incorporated herein by reference.

What is claimed:
 1. An aqueous pharmaceutical formulation comprising:(a) an anti-tumor necrosis factor alpha antibody comprising a lightchain variable region (LCVR) having a CDR3 domain comprising the aminoacid sequence of SEQ ID NO:3, a CDR2 domain comprising the amino acidsequence of SEQ ID NO:5, and a CDR1 domain comprising the amino acidsequence of SEQ ID NO: 7, and a heavy chain variable region (HCVR)having a CDR3 domain comprising the amino acid sequence of SEQ ID NO:4,a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 6, and aCDR1 domain comprising the amino acid sequence of SEQ ID NO:8, whereinthe concentration of the antibody is 50 to 200 mg/ml; (b) from 10-50 mMNaCl; (c) a surfactant; (d) a polyol; and (e) water; wherein theformulation does not comprise a buffering system, and wherein the pH ofthe formulation is from 4 to
 8. 2. The formulation of claim 1, whereinthe surfactant is a non-ionic surfactant.
 3. The formulation of claim 2,wherein the surfactant is a polysorbate.
 4. The formulation of claim 3,wherein the surfactant is polysorbate-20 or polysorbate-80.
 5. Theformulation of claim 1, wherein the polyol is a sugar or sugar alcohol.6. The formulation of claim 5, wherein the polyol is sucrose ormannitol.
 7. The formulation of claim 1, wherein the surfactant is anon-ionic surfactant, and wherein the polyol is a sugar or sugaralcohol.
 8. The formulation of claim 7, wherein the surfactant is apolysorbate.
 9. The formulation of claim 8, wherein the surfactant ispolysorbate-20 or polysorbate-80.
 10. The formulation of claim 7,wherein the polyol is sucrose or mannitol.
 11. The formulation of claim8, wherein the polyol is sucrose or mannitol.
 12. The formulation ofclaim 9, wherein the polyol is sucrose or mannitol.
 13. An aqueouspharmaceutical formulation comprising: (a) 100 mg/ml adalimumab; (b)from 10-50 mM NaCl; (c) a polysorbate; (d) a polyol, wherein the polyolis sucrose or mannitol; and (e) water; wherein the formulation does notcomprise a buffering system, and wherein the pH of the formulation isfrom 4 to
 8. 14. The formulation of claim 13, wherein the polysorbate ispolysorbate-20 or polysorbate-80.
 15. An aqueous pharmaceuticalformulation comprising: (a) 100 mg/ml adalimumab; (b) from 10-50 mMNaCl; (c) polysorbate-80; (d) a polyol, wherein the polyol is sucrose ormannitol; and (e) water; wherein the formulation does not comprise abuffering system, and wherein the pH of the formulation is from 4 to 6.16. The formulation of claim 15, wherein the pH is from 5 to
 6. 17. Theformulation of claim 15, wherein the polyol is sucrose.
 18. Theformulation of claim 15, wherein the polyol is mannitol.
 19. Theformulation of claim 16, wherein the polyol is sucrose.
 20. Theformulation of claim 16, wherein the polyol is mannitol.